Use of cdk9 and brd4 inhibitors to inhibit inflammation

ABSTRACT

Provided are methods for the combined use of cyclin-dependent kinase 9 (CDK9) inhibitors and bromodomain containing 4 (BRD4) inhibitors to reduce, inhibit and/or prevent cartilage degradation and systemic traumatic inflammation. A combination of CDK9 inhibitors and BRD4 inhibitors can be used to reduce, inhibit and/or prevent cartilage degradation and loss of cartilage viability during allograft storage. A combination of CDK9 inhibitors and BRD4 inhibitors can be used as a post-injury intervention treatment to reduce, inhibit and/or prevent the acute cellular responses that lead to future cartilage degradation and osteoarthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase under 35 U.S.C. §371 ofIntl. Appl. No. PCT/US2015/055394, filed on Oct. 13, 2015, which claimsthe benefit under 35 U.S.C. 119(e) of U.S. Provisional Appl. No.62/063,839, filed on Oct. 14, 2014, which are hereby incorporated hereinby reference in their entireties for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Grant No. AR063348awarded by the National Institutes of Health and Grant No. PRMRP ERAPR110507 awarded by the Department of Defense. The government hascertain rights in the invention.

FIELD

Provided are methods for the combined use of cyclin-dependent kinase 9(CDK9) inhibitors and bromodomain containing 4 (BRD4) inhibitors toreduce, inhibit and/or prevent cartilage degradation and systemictraumatic inflammation. A combination of CDK9 inhibitors and BRD4inhibitors can be used to reduce, inhibit and/or prevent cartilagedegradation and loss of cartilage viability during allograft storage. Acombination of CDK9 inhibitors and BRD4 inhibitors can be used as apost-injury intervention treatment to reduce, inhibit and/or prevent theacute cellular responses that lead to future cartilage degradation andosteoarthritis.

BACKGROUND

A host of pro-inflammatory and cellular stress induces inflammatoryresponse in chondrocytes, leading to upregulation of matrixmetalloproteinases (MMPs) and aggrecanases that degrade the cartilagematrix. Chronic deregulation of these catabolic pathways is suspected ofcausing osteoarthritis. Regardless of the sources of inflammation, thedownstream signals all converge on a common mechanism that activatestranscription of all primary response genes. This regulatory point iscontrolled by the transcription factor cyclin-dependent kinase 9 (CDK9)and its T-type cyclin partner. It was believed for many years that therate-limiting step in transcriptional activation is the recruitment oftranscription factors and RNA Polymerase II (Pol II) to gene promoters.However, recent studies on primary response genes have shown that intheir basal and unstimulated states, Pol II is already pre-assembled butis paused at the promoters (Hargreaves, et al., Cell 2009, 138:129-45;Zippo, et al., Cell 2009, 138:1122-36). The rapid activation of thesegenes is the result of signal-induced recruitment of CDK9 to thepromoters, where it phosphorylates Pol II. Phosphorylation by CDK9induces a conformational change that allows Pol II to enter possessiveelongation to efficiently transcribe full-length mRNAs (Zhou and Yik,MMBR 2006, 70(3): 646-659). Given that CDK9 controls a common mechanismof transcriptional activation of inducible genes, it is an effectivetarget for inhibiting the undesirable inflammatory responses fromdiverse cellular stress, such as sports-related injuries. The presentinvention is based, in part, on the discovery that pharmacological CDK9inhibitors, e.g., flavopiridol, and analogs and salts thereof, caneffectively suppress primary inflammatory genes in human articularchondrocytes in vitro. Effective suppression of inflammatory responsesallows for longer storage life for osteochondral explants used commonlyin cartilage repair, and also has therapeutic implications in preventingcartilage breakdown in post-traumatic osteoarthritis.

Severe combat trauma such as those received from explosive devices causedevastating damage to the human body, resulting in dramatic tissue loss,burns, orthopaedic injuries, and hemorrhagic shock. The reaction to suchsevere polytrauma can include a whole body response called systemicinflammatory response syndrome (SIRS). SIRS is the results of anexaggerated production of pro-inflammatory cytokines that illicit anoverwhelming inflammatory response throughout the entire body. While acontrolled local inflammation facilitates wound healing, theoverwhelming inflammatory response of SIRS significantly increases therisk of life-threatening events such as multiple organ dysfunction andfailure. Surgical treatment of injuries is often delayed afterpolytrauma due to the risk of SIRS-induced organ failure or death. Thisdelays recovery and increases associated medical costs. No pharmacologicagents effectively prevent the onset of SIRS or significantly improveits outcome; there is an urgent need to develop such drugs. Currentanti-inflammatory treatments are ineffective in part because they targetindividual cytokines and pathways, but in SIRS there are simply too manycytokines and pathways involved.

Since the problems of SIRS stem from the activation of the inflammatoryresponse, the conventional approaches to prevent SIRS have targeted oneor several of the many individual inflammatory signals or pathways. Theresults from these studies are mostly disappointing. Severe traumaticinjuries activate too many inflammatory signals and pathways, withsignificant cross talk between the pathways, for this one-by-oneapproach to be effective. The diverse inflammatory signals propagatethrough dozens of different cell surface receptors and an intricatenetwork of intracellular signaling pathways, which are then transmittedinto the cell nucleus for the activation of thousands of inflammatorymediator genes. To make matters worse, the inflammatory receptors andsignaling pathways have multiple levels of redundancy and cross talk.Therefore, no single agent that targets only a specific inflammatoryreceptor/mediator, signaling pathway, or cell type can prevent SIRS. Itis also impractical to formulate a drug cocktail that targets all knowninflammatory mediators and pathways at once. In addition, the initialexaggerated pro-inflammatory response in SIRS is activated rapidly aftertrauma; and therefore, the ideal anti-SIRS agents must also be able toact almost as quickly.

SUMMARY

In one aspect, methods of reducing, preventing, inhibiting, mitigatingand/or ameliorating cartilage degradation and/or chondrocyte death in asubject in need thereof are provided. In some embodiments, the methodscomprise co-administering to the subject an effective amount of aninhibitor of cyclin-dependent kinase 9 (CDK9) and an inhibitor ofbromodomain containing 4 (BRD4), thereby reducing, preventing,inhibiting, mitigating and/or ameliorating cartilage degradation and/orchondrocyte death in the subject. In a further aspect, methods ofreducing, preventing, inhibiting, mitigating and/or ameliorating theonset and/or progression of post-traumatic osteoarthritis in a subjectin need thereof are provided. In some embodiments, the methods compriseco-administering to the subject an effective amount of an inhibitor ofcyclin-dependent kinase 9 (CDK9) and an inhibitor of bromodomaincontaining 4 (BRD4), thereby reducing, preventing, inhibiting,mitigating and/or ameliorating post-traumatic osteoarthritis in thesubject. In varying embodiments, the subject has experienced a traumaticinjury to cartilage tissue. In varying embodiments, the traumatic injuryinduces acute overexpression of primary response genes. In varyingembodiments, the subject has undergone joint surgery. In another aspect,provided are methods of reducing, preventing, inhibiting, mitigatingand/or ameliorating severe polytrauma or systemic inflammatory responsesyndrome (SIRS) in a subject in need thereof In some embodiments, themethods comprise administering to the subject an effective amount of aninhibitor of cyclin-dependent kinase 9 (CDK9), thereby reducing,preventing, inhibiting, mitigating and/or ameliorating severe polytraumaor systemic inflammatory response syndrome (SIRS) in the subject. In arelated aspect, provided are methods of reducing, preventing,inhibiting, mitigating and/or ameliorating severe polytrauma or systemicinflammatory response syndrome (SIRS) in a subject in need thereof Insome embodiments the methods comprise co-administering to the subject aneffective amount of an inhibitor of cyclin-dependent kinase 9 (CDK9) andan inhibitor of bromodomain containing 4 (BRD4), thereby reducing,preventing, inhibiting, mitigating and/or ameliorating severe polytraumaor systemic inflammatory response syndrome (SIRS) in the subject. Insome embodiments, the subject may have experienced a traumatic injury,e.g., leading to acute systemic inflammation and to multiple organsystem failure. In varying embodiments, one or both of the inhibitor ofCDK9 and the inhibitor of BRD4 are initially co-administered during theacute response phase and reduce and/or inhibit transcriptionalactivation of one or more primary response genes after experiencingtraumatic injury. In some embodiments, the one or more primary responsegenes are selected from the group consisting of IL-1β, inducible nitricoxide synthase (iNOS), IL-6, TNF-α, MMP-1, MMP-3, MMP-9, MMP-13 andADAMTS4 (aggrecanase). In varying embodiments, one or both of theinhibitor of CDK9 and the inhibitor of BRD4 are initiallyco-administered within about 72 hours, about 48 hours, about 24 hours,e.g., within about 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1 or fewerhours, after experiencing traumatic injury. In varying embodiments, oneor both of the inhibitor of CDK9 and the inhibitor of BRD4 areadministered to the subject within about 10 days after damage or injury.In varying embodiments, one or both of the inhibitor of CDK9 and theinhibitor of BRD4 are administered over a course of 10 days. In varyingembodiments, one or both of the inhibitor of CDK9 and the inhibitor ofBRD4 are administered multiple times. In varying embodiments, one orboth of the inhibitor of CDK9 and the inhibitor of BRD4 are administereddaily for three consecutive days post-injury. In varying embodiments,the subject has undergone surgery to repair damaged cartilage tissue. Invarying embodiments, the subject has received an osteochondral explant.In varying embodiments, the osteochondral explant is a cartilageallograft. In varying embodiments, one or both of the inhibitor of CDK9and the inhibitor of BRD4 are co-administered concurrently with or priorto surgery. In varying embodiments, one or both of the inhibitor of CDK9and the inhibitor of BRD4 are initially co-administered within about 72hours, about 48 hours, about 24 hours, e.g., within about 22, 20, 18,16, 14, 12, 10, 8, 6, 4, 2, 1 or fewer hours, after surgery. In varyingembodiments, one or both of the inhibitor of CDK9 and the inhibitor ofBRD4 are co-administered systemically. In varying embodiments, one orboth of the inhibitor of CDK9 and the inhibitor of BRD4 areco-administered intravenously. In varying embodiments, one or both ofthe inhibitor of CDK9 and the inhibitor of BRD4 are co-administereddirectly to the site of injured cartilage tissue. In varyingembodiments, one or both of the inhibitor of CDK9 and the inhibitor ofBRD4 are delivered from a matrix. In varying embodiments, the inhibitorof CDK9 is a small organic compound. In varying embodiments, theinhibitor of CDK9 is flavopiridol. In varying embodiments, one of claims1 to 16, wherein the inhibitor of BRD4 is a small organic compound. Invarying embodiments, the inhibitor of BRD4 is selected from JQ1 andGSK525762A. In varying embodiments, one or both of the inhibitor of CDK9and the inhibitor of BRD4 are administered at a subtherapeutic ornon-efficacious dose for the individual inhibitors.

In another aspect, provided are methods of reducing, preventing orinhibiting degradation of an osteochondral explant and/or reducing,preventing or inhibiting chondrocyte death during storage. In someembodiments, the methods comprise storing the osteochondral explantand/or chondrocytes in a solution comprising an inhibitor ofcyclin-dependent kinase 9 (CDK9) and an inhibitor of bromodomaincontaining 4 (BRD4). In varying embodiments, the osteochondral explantis allograft cartilage. In varying embodiments, the inhibitor of CDK9 isa small organic compound. In varying embodiments, the inhibitor of CDK9is flavopiridol. In varying embodiments, the inhibitor of BRD4 is asmall organic compound. In varying embodiments, the inhibitor of BRD4 isselected from JQ1 and GSK525762A. In varying embodiments, theosteochondral explant is submerged in the solution comprising theinhibitor of CDK9 and the inhibitor of BRD4. In varying embodiments, thesolution comprises a sub-efficacious amount of one or both of theinhibitor of CDK9 and the inhibitor of BRD4.

In a further aspect, provided are compositions comprising anosteochondral explant in a solution comprising an inhibitor ofcyclin-dependent kinase 9 (CDK9) and an inhibitor of bromodomaincontaining 4 (BRD4). In varying embodiments, the osteochondral explantis allograft cartilage. In varying embodiments, the inhibitor of CDK9 isa small organic compound. In varying embodiments, the inhibitor of CDK9is flavopiridol. In varying embodiments, the inhibitor of BRD4 is asmall organic compound.

In varying embodiments, the inhibitor of BRD4 is selected from JQ1 andGSK525762A. In varying embodiments, the osteochondral explant issubmerged in the solution comprising the inhibitor of CDK9 and theinhibitor of BRD4. In varying embodiments, the solution comprises asub-efficacious amount of one or both of the inhibitor of CDK9 and theinhibitor of BRD4. In varying embodiments, the compositions are packagedin a kit.

DEFINITIONS

The term “cyclin-dependent kinase 9” or “CDK9” refers to a member of thecyclin-dependent protein kinase (CDK) family. CDK family members arehighly similar to the gene products of S. cerevisiae cdc28, and S. pombecdc2, and known as important cell cycle regulators. CDK9 was found to bea component of the multiprotein complex TAK/P-TEFb, which is anelongation factor for RNA polymerase II-directed transcription andfunctions by phosphorylating the C-terminal domain of the largestsubunit of RNA polymerase II. CDK9 forms a complex with and is regulatedby its regulatory subunit cyclin T or cyclin K. HIV-1 Tat protein wasfound to interact with this protein and cyclin T. Structurally, “CDK9”refers to nucleic acids and polypeptide polymorphic variants, alleles,mutants, and interspecies homologs that: (1) have an amino acid sequencethat has greater than about 90% amino acid sequence identity, forexample, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater aminoacid sequence identity, preferably over a region of at least about 25,50, 100, 200, 400, or more amino acids, or over the full-length, to anamino acid sequence encoded by a CDK9 nucleic acid (see, e.g., GenBankAccession No. NM_001261.3); (2) bind to antibodies, e.g., polyclonalantibodies, raised against an immunogen comprising an amino acidsequence of a CDK9 polypeptide (e.g., GenBank Accession No. NP001252.1); or an amino acid sequence encoded by a CDK9 nucleic acid(e.g., CDK9 polynucleotides described herein), and conservativelymodified variants thereof; (3) specifically hybridize under stringenthybridization conditions to an anti-sense strand corresponding to anucleic acid sequence encoding a CDK9 protein, and conservativelymodified variants thereof; (4) have a nucleic acid sequence that hasgreater than about 90%, preferably greater than about 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide sequence identity,preferably over a region of at least about 25, 50, 100, 200, 500, 1000,2000 or more nucleotides, or over the full-length, to a CDK9 nucleicacid (e.g., CDK9 polynucleotides, as described herein, and CDK9polynucleotides that encode CDK9 polypeptides, as described herein).Based on the knowledge of CDK9 homologs, those of skill can readilydetermine residue positions that are more tolerant to substitution. Forexample, amino acid residues conserved amongst species are less tolerantof substitution or deletion. Similarly, amino acid residues that are notconserved amongst species are more tolerant of substitution or deletion,while retaining the function of the CDK9 protein.

The term “bromodomain containing 4” or “BRD4” refers to a homologue ofthe murine protein MCAP, which associates with chromosomes duringmitosis, and to the human RING3 protein, a serine/threonine kinase. BRD4contains two bromodomains, a conserved sequence motif which may beinvolved in chromatin targeting. Structurally, “BRD4” refers to nucleicacids and polypeptide polymorphic variants, alleles, mutants, andinterspecies homologs that: (1) have an amino acid sequence that hasgreater than about 90% amino acid sequence identity, for example, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity, preferably over a region of at least about 25, 50, 100, 200,400, or more amino acids, or over the full-length, to an amino acidsequence encoded by a BRD4 nucleic acid (see, e.g., GenBank AccessionNos. NM_014299.2 (short transcript variant) and NM_058243.2 (longtranscript variant)); (2) bind to antibodies, e.g., polyclonalantibodies, raised against an immunogen comprising an amino acidsequence of a BRD4 polypeptide (e.g., GenBank Accession Nos. NP_055114.1(short transcript variant) and NP_490597.1 (long transcript variant));or an amino acid sequence encoded by a BRD4 nucleic acid (e.g., BRD4polynucleotides described herein), and conservatively modified variantsthereof; (3) specifically hybridize under stringent hybridizationconditions to an anti-sense strand corresponding to a nucleic acidsequence encoding a BRD4 protein, and conservatively modified variantsthereof; (4) have a nucleic acid sequence that has greater than about90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or higher nucleotide sequence identity, preferably over aregion of at least about 25, 50, 100, 200, 500, 1000, 2000 or morenucleotides, or over the full-length, to a BRD4 nucleic acid (e.g., BRD4polynucleotides, as described herein, and BRD4 polynucleotides thatencode BRD4 polypeptides, as described herein). Based on the knowledgeof BRD4 homologs, those of skill can readily determine residue positionsthat are more tolerant to substitution. For example, amino acid residuesconserved amongst species are less tolerant of substitution or deletion.Similarly, amino acid residues that are not conserved amongst speciesare more tolerant of substitution or deletion, while retaining thefunction of the BRD4 protein.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, a-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.

Such analogs have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions in a mannersimilar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles .

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine I, Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and-   7) Serine (S), Threonine (T)    (see, e.g., Creighton, Proteins (1984)).

The terms “identical” or percent “identity,” and variants thereof in thecontext of two or more polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a specified percentage of aminoacid residues or nucleotides that are the same (i.e., at least 60%identity, optionally at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity over a specified region (or the wholereference sequence when not specified)), when compared and aligned formaximum correspondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. The present invention providespolypeptides substantially identical to CDK9, as described herein.Optionally, the identity exists over a region that is at least about 50amino acids in length, or more preferably over a region that is 100 to500 or 1000 or more amino acids in length, or over the full-length ofthe sequence.

The terms “similarity,” or “percent similarity,” and variants thereof inthe context of two or more polypeptide sequences, refer to two or moresequences or subsequences that have a specified percentage of amino acidresidues that are either the same or similar as defined in the 8conservative amino acid substitutions defined above (i.e., 60%,optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%similar over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Sequences having less than 100%similarity but that have at least one of the specified percentages aresaid to be “substantially similar.” Optionally, this identity existsover a region that is at least about 50 amino acids in length, or morepreferably over a region that is at least about 100 to 500 or 1000 ormore amino acids in length, or over the full-length of the sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

The term “comparison window”, and variants thereof, includes referenceto a segment of any one of the number of contiguous positions selectedfrom the group consisting of from 20 to 600, usually about 50 to about200, more usually about 100 to about 150 in which a sequence may becompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. Methods ofalignment of sequences for comparison are well known in the art. Optimalalignment of sequences for comparison can also be conducted by the localhomology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981),by the homology alignment algorithm of Needle man and Wunsch J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson andLipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis.), Karlin and Altschul Proc.Natl. Acad. Sci. (U.S.A.) 87:2264-2268(1990), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

Examples of an algorithm that is suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information (onthe internet at ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. StandardBLAST algorithm parameters have an expected threshold of 10 (accordingto the stochastic model of Karlin and Altschul (PNAS,87:2264-2268(1990)); a word size of 28; reward and penalty of 1/−2 (aratio of 0.5, or 1/−2, is used for sequences that are 95% conserved);and a linear GAP cost.

The term “effective amount” refers to an amount (here of an inhibitor ofCDK9) which provides either subjective relief of a symptom(s) or anobjectively identifiable improvement as noted by the clinician or otherqualified observer. Determination of an effective amount is well withinthe capability of those skilled in the art, especially in light of thedetailed disclosure provided herein. Generally, an efficacious oreffective amount of a combination of one or more polypeptides of thepresent invention is determined by first administering a low dose orsmall amount of a polypeptide or composition and then incrementallyincreasing the administered dose or dosages, adding a second or thirdmedication as needed, until a desired effect of is observed in thetreated subject with minimal or no toxic side effects. Applicablemethods for determining an appropriate dose and dosing schedule foradministration of a combination of the present invention are described,for example, in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 12th Edition, 2010, supra; in a Physicians' Desk Reference(PDR), 68^(th) Edition, 2014, PDR Network; in Remington: The Science andPractice of Pharmacy, 21^(st) Ed., 2006, supra; and in Martindale: TheComplete Drug Reference, Sweetman, 2005, London: Pharmaceutical Press.,and in Martindale, Martindale: The Extra Pharmacopoeia, 31st Edition.,1996, Amer Pharmaceutical Assn, each of which are hereby incorporatedherein by reference.

“Subtherapeutic dose or amount” or “non-efficacious does or amount”interchangeably refer to a dose of a pharmacologically active agent(s),either as an administered dose of pharmacologically active agent, oractual level of pharmacologically active agent in a subject thatfunctionally is insufficient to elicit the intended pharmacologicaleffect in itself (e.g., to obtain anti-inflammatory effects), or thatquantitatively is less than the established therapeutic dose for thatparticular pharmacological agent (e.g., as published in a referenceconsulted by a person of skill, for example, doses for a pharmacologicalagent published in the Physicians' Desk Reference, 68th Ed., 2014, PDRNetwork or Brunton, et al., Goodman & Gilman's The Pharmacological Basisof Therapeutics, 12th edition, 2010, McGraw-Hill Professional). A“subtherapeutic dose” can be defined in relative terms (i.e., as apercentage amount (less than 100%) of the amount of pharmacologicallyactive agent conventionally administered). For example, a subtherapeuticdose amount can be about 1% to about 75% of the amount ofpharmacologically active agent conventionally administered. In someembodiments, a subtherapeutic dose can be about 75%, 50%, 30%, 25%, 20%,10% or less, than the amount of pharmacologically active agentconventionally administered.

The terms “treating” and “treatment” and variants thereof refer topromoting healing, delaying the onset of, retarding or reversing theprogress of, alleviating or preventing either the disease or conditionto which the term applies (e.g., cartilage degradation), or one or moresymptoms of such disease or condition. Treating and treatment encompassboth therapeutic and prophylactic treatment regimens.

The terms “subject,” “patient,” or “individual” interchangeably refer toany mammal, for example, humans and non-human primates, domestic mammals(e.g., canine, feline), agricultural mammals (e.g., bovine, equine,ovine, porcine) and laboratory mammals (e.g., mouse, rat, rabbit,hamster).

As used herein, “administering” refers to local and systemicadministration, e.g., including enteral, parenteral, pulmonary, andtopical/transdermal administration. Routes of administration forcompounds (e.g., inhibitors of CDK9, e.g., flavopiridol, and analogs andsalts thereof) that find use in the methods described herein include,e.g., oral (per os (P.O.)) administration, nasal or inhalationadministration, administration as a suppository, topical contact,transdermal delivery (e.g., via a transdermal patch), intrathecal (IT)administration, intravenous (“iv”) administration, intraperitoneal(“ip”) administration, intramuscular (“im”) administration,intralesional administration, or subcutaneous (“sc”) administration, orthe implantation of a slow-release device e.g., a mini-osmotic pump, adepot formulation, etc., to a subject. Administration can be by anyroute including parenteral and transmucosal (e.g., oral, nasal, vaginal,rectal, or transdermal). Parenteral administration includes, e.g.,intravenous, intramuscular, intra-arterial, intradermal, subcutaneous,intraperitoneal, intraventricular, ionophoretic and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The terms “systemic administration” and “systemically administered”refer to a method of administering a compound or composition to a mammalso that the compound or composition is delivered to sites in the body,including the targeted site of pharmaceutical action, via thecirculatory system. Systemic administration includes, but is not limitedto, oral, intranasal, rectal and parenteral (e.g., other than throughthe alimentary tract, such as intramuscular, intravenous,intra-arterial, transdermal and subcutaneous) administration.

The term “co-administering” or “concurrent administration”, when used,for example with respect to the compounds (e.g., inhibitors of CDK9,e.g., flavopiridol, and analogs and salts thereof) and/or analogsthereof and another active agent, refers to administration of thecompound and/or analogs and the active agent such that both cansimultaneously achieve a physiological effect. The two agents, however,need not be administered together. In certain embodiments,administration of one agent can precede administration of the other.Simultaneous physiological effect need not necessarily require presenceof both agents in the circulation at the same time. However, in certainembodiments, co-administering typically results in both agents beingsimultaneously present in the body (e.g,. in the plasma) at asignificant fraction (e.g., 20% or greater, preferably 30% or 40% orgreater, more preferably 50% or 60% or greater, most preferably 70% or80% or 90% or greater) of their maximum serum concentration for anygiven dose. For example, in various embodiments, an inhibitor of CDK9 isco-administered with protein complexes or protein scaffolds comprisingone or more monomers of cartilage oligomeric matrix protein (COMP) boundto one or more growth factors, as described in co-owned and co-pendingInternational Appl. No. PCT/US2011/051610.

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the agent(s)/compound(s) at issue to the subject. Causing to beadministered can involve diagnosis and/or determination of anappropriate therapeutic or prophylactic regimen, and/or prescribingparticular agent(s)/compounds for a subject. Such prescribing caninclude, for example, drafting a prescription form, annotating a medicalrecord, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an approach for anti-inflammatory therapy: Cdk9controls the rate-limiting step for activation of primary inflammatoryresponse genes. There are many different inflammatory stimuli, forexample, IL-1β, LPS, and TNF activate their respective cell surfacereceptors and signal through different intracellular mediators/pathways,which ultimately converge on Cdk9-dependent activation of RNA pol II fortranscription of most, if not all, primary inflammatory response genes.Therefore, targeting Cdk9's kinase activity, or its recruitment topromoter via Brd4, is ideal for effectively blocking all inflammatorygenes activation regardless of the inflammatory source. Flavopiridol andJQ1 are small molecules Cdk9 and Brd4 inhibitors, respectively.

FIGS. 2A-B. FIG. 2A illustrates the structure of Flavopiridol, a smallmolecule (MW=402) kinase inhibitor with a Kd of 3-6 nM for Cdk9. FIG. 2Billustrates the structure of JQ1, a small molecule Brd4 inhibitor(MW=456) that prevents Brd4 from binding acetylated histones andrecruiting Cdk9 to promoters.

FIG. 3 illustrates determination of the minimal combined dosage forFlavopiridol and JQ1 to achieve complete repression of IL-1β response.The red curve indicates the combined drug dose at which the IL-1βresponse is completely repressed. Combined treatment reduced therequired dosage of Flavopiridol and JQ1 to 60 and 250 nM, about 20-25%of the required dose of each drug individually to achieve 100%repression. Results were generated from healthy human chondrocytes from4 individual donors, and DOE performed using JMP 11.0 statisticalsoftware.

FIGS. 4A-B illustrate CDK9/BRD4 inhibitors repress chondrocyte responseto multiple catabolic cytokines. Induction of iNOS and Cox2 byinflammatory cytokines (IL-1β and TNF) requires BRD4 and CDK9 activity.mRNA expression of the cytokine-treated groups were normalized to 100%(blue bars) and compared to groups with cytokine plus CDK9/BRD4inhibitors (***:p<0.001, *:p<0.05).

FIGS. 5A-C illustrate that Cdk9 inhibition, alone and with concurrentBrd4 inhibition blocks multiple upstream inflammatory signals. A) Cdk9inhibition by Flavopiridol is effective against multiple inflammatorystimuli. Human primary chondrocytes (n=3 donors) were treated with 3different inflammatory stimuli (10 ng/ml of either IL-1β, LPS, or TNF)with or without 300 nM Flavopiridol for 5 hours. The inducible nitricoxide synthase (iNOS) mRNA was quantified by real-time PCR as arepresentative of primary response genes. iNOS induction by eachstimulus alone was arbitrarily set to 100% (first bar) and compared tothe respective samples co-treated with Flavopiridol. Panel C)demonstrates that both Flavopiridol and JQ1 suppress the inflammatoryresponse induced by IL-6. B) siRNA-mediated depletion of Cdk9 repressesiNOS induction. Chondrocytes were transduced with lentiviral particlesharboring siRNA against Cdk9 or GFP as control.

After 5 days, cells were treated with IL-1β for 5 hours and harvestedfor Western (inset) and iNOS mRNA analysis. For all the aboveexperiments, each data point was the mean +/−standard deviation from 3different human donors. (*/**p<0.05).

FIGS. 6A-B illustrate that Cdk9 inhibition protects cartilage explantsfrom mechanical injury-induced inflammation and apoptosis. Bovinecartilage explants (6 mm diameter×3 mm height, n=6) were subjected to asingle load of compression of 30% strain/sec, then placed into mediawith or without 300 nM Flavopiridol. A) The expression of IL-6 mRNA overa period of 24 hours post-injury was determined by qPCR. (C=control,I=mechanically injured). Injury induced IL-6 mRNA but the induction wasprevented by Cdk9 inhibitors. B) Cdk9 inhibition prevents injury inducedapoptosis in cartilage explants. Mechanically injured cartilage explants(n=3 donors) were cultured with or with Flavopiridol and processed forhistology. Apoptotic cells were detected with TUNEL staining (DeadEndFluorometric TUNEL system, Promega) and calculated as percentage oftotal cells (DAPI stain). Injury increased apoptotic cell population butthe effect was reduced by Cdk9 inhibition in all time points tested(*p<0.05).

FIGS. 7A-B illustrate that Cdk9 inhibition globally and effectivelysuppresses activation of downstream primary inflammatory response genes.A) Primary human chondrocytes (n=2) were treated with 10 ng/ml IL-1β,with or without Flavopiridol (F) and/or JQ1 (J) for 5 hours. The entiretranscriptomes were analyzed by microarrays (GeneChip Human Gene 2.0 STArray, Affymetrix). Among the expressed genes, 873 were induced>1.5-foldby IL-1β (compared lanes 1 to 5). The normalized intensities of each ofthese 873 genes were shown as heat map. Flavopiridol and/or JQ1treatment suppressed the majority of these genes. B) Highly effectivesuppression of IL-1β-induced genes by Cdk9 inhibitors. The numbers ofinducible genes suppressed by Cdk9 inhibitors were categorized into fourquadrants according to the percentage of repression from their maximuminduction. Induction of most genes was strongly suppressed by Cdk9inhibitors. Co-treatment with both Flavopiridol and JQ1 showed a maximalsynergistic effect at about 20-25% of the original dosages (250:60 nM),demonstrating the benefits of combined drug treatments in reducing totaldosage and potential off-target effects.

FIGS. 8A-B illustrate Catabolic genes in chondrocytes were repressed byCDK9 and BRD4 inhibitors. Induction of matrix-degrading proteases byinflammatory cytokines requires BRD4 and CDK9 activity. Inhibition ofBRD4, CDK9 activity almost completely repressed cytokine-inducedexpression of MMPs and ADAMTS4.

FIGS. 9A-H illustrate that CDK9 inhibition prevents injury-inducedupregulation of pro-inflammatory cytokine and catabolic genes. A)Schematic representation of the cartilage explant injury and drugtreatment, and the subsequent tests. B-F) Suppression ofpro-inflammatory cytokine and catabolic enzyme genes by Flavopiridol incartilage explants (C=control uninjured, I=injured) within 24 hourspost-injury, G-H) Flavopiridol does not affect the expression of theanabolic genes aggrecan and collagen 2A. All values were themean+/−standard deviation obtained from n=6 individual donors (*p<0.05).

FIGS. 10A-C illustrate that CDK9 inhibition reduces mRNA expression ofapoptotic mediators. Flavopiridol suppresses mRNA expression of thepro-apoptotic genes p53, Bcl-2, and PTEN. All values were themean+/−standard deviation obtained from n=6 individual donors (*p<0.05).

FIGS. 11A-B illustrate CDK9 inhibition rescues chondrocyte frominjury-induced apoptosis. A) Flavopiridol treatment prevents chondrocyteapoptosis. At the indicated times, cartilage explants were processed forhistological examination and TUNEL staining. Injury increased apoptosisin cartilage explants but Flavopiridol reduced the apoptotic cellpopulation. Results were the mean+/−standard deviation obtained from n=3individual donors (*p<0.05). B) Flavopiridol enhances chondrocytesurvival. At 5-days post-injury, cartilage explants were stained withLIVE/DEAD viability stain. Injury decreased cell viability but the cellswere rescued by Flavopiridol. Results were the mean+/−standard deviationobtained from n=6 individual donors (*p<0.05).

FIG. 12 illustrates that protection of cartilage from injury-inducedmatrix degradation. Mechanically injured cartilage explants were treatedfor 5 days with Flavopiridol. The amount of GAG released into the mediumwas measured by dimethylmethylene blue dye binding assay. Results werenormalized to the wet weight of the explants. Injury caused cartilagedegradation, as indicated by increased GAG release. In the presence of300 nM Flavopiridol, levels of GAG release returned to baseline. Resultswere the mean+/−standard deviation obtained from n=6 individual donors(*p<0.05).

FIGS. 13A-C illustrate that CDK9 inhibition preserves the mechanicalproperties of injured cartilage. Cartilage explants were cultured for 4weeks post-injury. The cartilage compressive properties were assessed bystress-relaxation testing in unconfined compression. The 10% relaxationand instantaneous moduli, and coefficient of viscosity are shown.Flavopiridol treatment enhanced the compressive properties of both theinjured and uninjured explants. Results were the mean+/−standarddeviation obtained from n=6 individual donors. Within each chart, meansthat do not share a letter are significantly different from each other(P<0.05).

FIG. 14 illustrates that CDK9 inhibition reduces injury-induced loss ofcartilage stiffness. Right knees of mice were injured (n=6) as describedin Yik, et al., Arthritis Rheumatol. (2014) 66(6):1537-46. Left kneesserved as uninjured controls. Flavopiridol (2.5 mg/kg) and JQ1 (17mg/kg) were administered daily by intraperitoneal injection for threeconsecutive days post-injury. At day 7, animal knees were harvested andthe cartilage stiffness at the lateral (L) and medial (M) femoralcondyles were determined with Atomic Force Microscopy as described (Liet al., Journal of Biomechanics. (2015) 48(8):1364-70). Injury induces aloss of cartilage stiffness but the loss is reduced buy the combineddrug treatment. Non-invasive mouse models of post-traumaticosteoarthritis are also described in, e.g., Christiansen, et al.,Osteoarthritis Cartilage. (2015) 23(10):1627-38.

DETAILED DESCRIPTION 1. Introduction

Provided are methods based, in part, on the discovery that combinedinhibition of CDK9 and BRD4 activity finds use to prevent, reduce,and/or mitigate polytrauma or SIRS, preserve cartilage after injury(e.g., traumatic injury and/or surgical injury) and during storage(e.g., allograft storage). Any combination of CDK9 and BRD4 inhibitorsknown in the art can be used in the present methods, includinginhibitory nucleic acids and inhibitory compounds (e.g., flavopiridol,JQ1, GSK525762A, and analogs and salts thereof).

Many different stimuli can induce inflammation, and several of these arebeing investigated individually as arthritis drugs (e.g., IL-1antagonists, TNF antagonists, anti-oxidants). The focus has been oninhibition of the pathway so that transcription of response genes doesnot occur. None of these existing investigations have addressed theprocess of transcription. The present invention is based, in part, onthe discovery that all of these pathways converge on the combinedactivation of CDK9 and BRD4 for the transcriptional elongation of theprimary response genes. Inhibition of the transcriptional elongation bycombined inhibition of CDK9 and BRD4 is limited to the primary responseinflammatory genes, and combined CDK9 and BRD4 inhibition does notaffect transcription of housekeeping genes, and therefore is notdetrimental to cells or tissues in the short term. The combinedadvantage of CDK9 and BRD4 inhibition is that it reduces transcriptionalelongation of inflammatory genes from all inflammatory stimuli. Invarious embodiments, CDK9 and BRD4 can be specifically and reversiblyinhibited, e.g., with small-molecule drugs (e.g., flavopiridol and otherknown CDK9 inhibitors; JQ1, GSK525762A, and other known BRD4 inhibitors,and analogs and salts thereof) and/or inhibitory nucleic acids (e.g.,siRNA, miRNA, antisense RNA).

Symptomatic osteoarthritis (OA) can be defined as the end-stage failureof load bearing joints at the organ level (1). While the etiology of OAremains incompletely understood, it is well established that jointinjuries often progress to OA over time (2). High-energy joint traumasthat cause intra-articular fractures often result in the rapiddevelopment of joint degradation and post-traumatic osteoarthritis(PTOA) (3). Even lower-energy traumas to the joint, which are much morecommon, will initiate slowly progressing cartilage and joint degradationthat results in symptomatic PTOA many years later (2). As an example,from a total of 900,000 knee injuries annually in the United States(4),the American Academy of Orthopaedic Surgeons estimates 200,000 injuriesof the anterior cruciate ligament (ACL) in the general population (5),including 2500 to 3000 ACL reconstructions in military patients (6).Strikingly, approximately 50% of these ACL injury patients will developknee PTOA after a 10- to 20-year asymptomatic lag phase (7).

NFL retirees under the age of 50 are five times more likely to havearthritis than comparable men in the general population, approximately80% of retirees report having joint pain lasting most of the day, andover 23% of NFL retirees over 50 years of age have had a jointreplacement.

Despite a lack of joint pain during the asymptomatic lag phase,progressive deterioration of bone and cartilage begins to develop soonafter traumatic joint injury. In OA of the knee or hip joints, theasymptomatic cartilage degeneration phase can last many years or evendecades (8). By the time arthritic joints become painful, there is oftenwidespread cartilage damage with areas of complete cartilage loss. As aresult of the extended painless “pre-OA” condition, the typical OApatient is seen in the clinic only after extensive joint damage hasalready occurred. At these late stages, treatment of the underlyingcauses of joint degeneration is no longer possible and the damage hasbecome irreversible. Current OA treatments address the associated jointpain, but do not improve joint function or alter the underlyingpathology. When these palliative treatments eventually fail, invasivesurgical joint replacement is the only remaining treatment for pain-freeambulation. Although there is abundant evidence that joint traumas suchas anterior cruciate ligament (ACL) tears will ultimately lead to OA(9-11), current clinical treatment does nothing at the time of theseinjuries to prevent the future onset of OA. Currently, clinicaltreatment is aimed at reducing the immediate pain and swelling in thejoint and restoring normal joint movement. The most commonrecommendations are to apply ice, gently compress the joint with anelastic bandage, and take pain medications such as aspirin,acetaminophen, or ibuprofen. Importantly, these treatments do notaddress the initiation of OA. The incidence of OA is independent ofwhether patients undergo surgical reconstruction of the ACL (7,12),suggesting that the injury event, in addition to the chronic jointinstability, has a causative role in OA pathogenesis.

The mechanical damage that a joint experiences during an impact hasimmediate effects on the tissues: cell death and physical damage to thejoint tissues occur within milliseconds of impact. The immediatemechanical damage then triggers an acute cellular response, which occurswithin a time-scale of minutes to hours (13). The acute response phaseis characterized by the release of inflammatory mediators from theinjured joint tissues, including IL-1, IL-6, iNOS, and TNF-a (13,14).This causes the transcriptional activation of primary response genes (orinflammatory genes), and leads to increased production of matrixdegrading enzymes such as MMPs, collagenases, aggrecanases, andcathepsins. The enzymatic degradation of matrix contributes to OA via acascade of destructive events, including:

-   -   (1) reducing the stiffness and elasticity of cartilage, thus        increasing the mechanical stresses on chondrocytes,    -   (2) increasing the hydraulic permeability of cartilage, leading        to loss of interstitial fluid and increased diffusion of solutes        (i.e. degradative enzymes, proteoglycans),    -   (3) increasing the accessibility of remaining cartilage matrix        structures to enzymatic digestion,    -   (4) thickening of the subchondral bone plate,    -   (5) structural changes to the trabecular bone, and    -   (6) formation of osteophytes and heterotopic bone (8).

We believe that a window for therapeutic intervention exists shortlyafter injury, during which attenuating the acute cellular responsedecreases production of matrix degrading enzymes and thus decreases thelikelihood of developing post-traumatic osteoarthritis (PTOA).

One strategy to attenuate the systemic inflammatory response would be totarget a common mechanism that controls these multiple inflammatoryfactors. Herein, we have identified a single conserved mechanism, in thecell nucleus, that controls the transcriptional elongation of allprimary response genes. Inhibition of this common mechanism is possiblewith small molecule drugs already in clinical trials. Applying thiscompletely new therapeutic strategy to severe trauma patients has greatpotential to save lives and decrease the medical costs by reducing theeffects of SIRS, allowing the trauma team to more quickly stabilize thepatient and treat injuries.

The transcriptional activation of primary response genes is an importantstep of the acute cellular response to injury. Transcriptionalactivation of primary response genes occurs in a timeframe of minutes tohours after the injury event. The majority of the primary response genesare ‘primed’ for transcription at a moment's notice, with thetranscription complex already assembled on the promoters and the RNApolymerase complex stalled just before entering the transcriptionelongation stage. In a recent Cell paper, Hargreaves et al elegantlydemonstrated that the rate-limiting step in transcriptional elongationof primary response genes is the recruitment of cyclin-dependentkinase-9 (CDK9) (15). In the case of inflammatory gene transcription,CDK9 is recruited to the transcription complex by NFKB (16,17).Importantly, CDK9 kinase activity is required for transcription of theprimary response inflammatory genes to proceed, and this mechanism ofregulation is conserved amongst primary response genes (18). Thus,combined inhibition of CDK9 kinase activity and BRD4 activity representsa new molecular target to inhibit the acute inflammatory response afterjoint injury.

After severe trauma, a plethora of conditions and sources can causesystemic inflammatory response syndrome (SIRS). Conventionalanti-inflammatory drugs are ineffective against SIRS, because they onlytarget a specific branch of the upstream inflammatory signalingnetworks, or one of the hundreds of inflammatory mediator genes (seeFIG. 1). For example, corticosteroids, TLR4 antagonist, TNF and IL-1receptor antagonists, antibradykinin, platelet activating factorreceptor antagonists, and anticoagulants (antithrombin III) have allbeen studied without showing significant benefits in clinical trials.The present methods concurrently inhibit Cdk9 and Brd4 for suppressingsystemic inflammation. By simultaneously inhibiting Cdk9 and Brd4, weblock the rate-limiting step and bottle-neck for activation of allprimary inflammatory response genes. The advantages of our approach aretwo-fold. First, it is effective against all upstream inflammatorystimuli regardless of their initiating signaling pathways. Second, it isefficient in suppressing the expression of most downstream inflammatoryresponse genes.

Therefore, blocking Cdk9 will block the full spectrum of inflammatoryresponses and represent a far more superior approach for modulatingsystemic inflammation after severe trauma.

2. Subjects Who May Benefit

Subjects who can benefit from a regime of combined one or more CDK9inhibitors and one or more BRD4 inhibitors generally have experienced orimminently will experience a traumatic injury resulting in acutesystemic inflammation, and/or an injury to cartilage tissue. In someembodiments, the subject may have experienced an injury (e.g., atraumatic injury), e.g., that damages cartilage tissue. In someembodiments, the subject may have experienced a traumatic injury, e.g.,leading to acute systemic inflammation and possibly to multiple organsystem failure. The subject may also undergo or have undergone surgery,e.g., to repair damaged cartilage tissue and/or to receive anosteochondral explant.

In various embodiments, a regime of combined one or more CDK9 inhibitorsand one or more BRD4 inhibitors is administered to the subject withinabout 10 days after damage or injury to cartilage tissue, for example,within about 9, 8, 7, 6, 5, 4, 3, 2 or 1 days after damage or injury tocartilage tissue. In various embodiments, a regime of combined one ormore CDK9 inhibitors and one or more BRD4 inhibitors are initiallyadministered to the subject within about 72 hours, about 48 hours orabout 24 hours after damage or injury to cartilage tissue orexperiencing trauma, for example, within about 22, 20, 18, 16, 14, 12,10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or fewer, hours after damage or injury tocartilage tissue or experiencing trauma.

3. Inhibitors of CDK9 and BRD4

Generally, the activity of a CDK9, e.g., a polypeptide having at least80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an amino acidsequence of NP_001252.1, is inhibited or reduced, thereby preventing,reducing, delaying or inhibiting degradation of cartilage and/or onsetor progression of post-traumatic osteoarthritis.

a. Small Organic Compounds

i. CDK9 Inhibitors

CDK9 is a member of the cyclin-dependent kinase family, and mostproteins in this family regulate cell-cycle progression. Over the last 2decades there has been intense research into CDK inhibitors asanti-proliferative agents that arrest cell cycle progression in cancers,and numerous CDK inhibitors are in phase II and III clinical trials(19,20). CDK9, unlike most CDK proteins that regulate cell cycleprogression, is mainly thought to regulate RNA synthesis andtranscriptional elongation (21). There are small-molecule inhibitorswith relatively good specificity for CDK9, including flavopiridol, andanalogs and salts thereof. Commercial preparations of flavopiridol arecalled Alvocidib The IUPAC name for flavopiridol is2-(2-chlorophenyl)-5, 7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone). The structure offlavopiridol is shown below.

Flavopiridol inhibits CDK9 kinase activity by a high affinity (Kd=3 nMto 6 nM) interaction with the ATP-binding pocket of CDK9 (22,23).Inhibition of CDK9 kinase activity prevents transcriptional activationof primary response genes by preventing transcriptional elongation (15).Systemic administration of flavopiridol is well tolerated, and clinicaltrials with flavopiridol are successful in treating refractory chroniclymphocytic leukemia (24-26). Recently, Sekine et al have takenadvantage of the anti-proliferative effects of flavopiridol in mousemodels of rheumatoid arthritis (RA). They demonstrated that flavopiridolreduced synovial hyperplasia and effectively prevented rheumatoidarthritis (27). The anti-arthritic effect was reversible; whenflavopiridol treatment was stopped, synovial hyperplasia resumed and RAprogressed rapidly.

Other CDK inhibitors that can find use include without limitation, e.g.,4-(3,5-Diamino-1H-pyrazol-4-ylazo)-phenol (Calbiochem Catalog No.238811),2-(Pyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridinone,PHA-767491 (Calbiochem Catalog No. 217707), and those described, e.g.,in International Publication Nos. WO 2012/101066 (pyridine biaryl aminecompounds); WO 2012/101065 (pyrimidine biaryl amine compounds); WO2012/101064 (N-acyl pyrimidine biaryl compounds); WO 2012/101063 (N-acylpyridine biaryl compounds); WO 2012/066070 (3-(aminoaryl)-pyridinecompounds); WO 2012/066065 (phenyl-heteroaryl amine compounds); WO2011/012661 (pyridine and pyrazine derivatives); WO 2011/077171(4-phenylamino-pyrimidine derivatives); WO 2010/020675(pyrrolopyrimidine compounds);

WO 2008/079933 (heteroaryl-heteroaryl compounds); WO 2007/117653(CDK9-PI3K-AKT inhibitors); WO 2006/024858(4-arylazo-3,5-diamino-pyrazole compounds); WO 2006/021803 (purine andpyrimidine CDK inhibitors) and in U.S. Patent Publication Nos.2012/0225899; 2012/0196855; 2012/0142680; 2010/0160350; 2010/0249149;2010/0076000; 2010/0035870; 2010/0003246; 2009/0325983; 2009/0318446;

2009/0318441; 2009/0270427; 2009/0258886; 2009/0215805; 2009/0215805;2009/0137572; 2008/0125404; 2007/0275963; 2007/0225270; 2007/0072882;2007/0021452; 2007/0021419; and 2006/0264628, all of which are herebyincorporated herein by reference in their entirety for all purposes.

ii. BRD4 Inhibitors

In addition to directly targeting the kinase activity of Cdk9 tosuppress inflammation, a new class of small molecule inhibitors thatblocks its recruitment to activated gene promoters by the bromodomainprotein Brd4 was recently discovered as an effective Cdk9 inhibitors(Filippakopoulos, et al., (2010) Nature 468, 1067-1073). The compoundJQ1 (FIG. 2 and below) is a bromodomain inhibitor that prevents Brd4from binding acetylated histones at the chromosome near the promoters ofactivated primary response genes. Thus, JQ1 indirectly interferes withCdk9 recruitment by Brd4 and has shown promising potential as abroad-spectrum anti-inflammatory agent in animal models (Belkina, etal., (2013) J Immunol 190, 3670-3678). In our data herein, wedemonstrate strong synergy between Cdk9 and Brd4 inhibitors in reducingand preventing inflammatory mediators.

Other BRD4 and bromodomain inhibitors that can find use include withoutlimitation, e.g., those described in U.S. Patent Publication Nos.2014/0296246, 2014/0296243, 2014/0243322, 2014/0243321, 2014/0243286,2014/0044770, 2012/0208800 and 2012/0157428; and in Intl. PublicationNos. WO 2014/143768, WO 2014/160873, WO 2014/128067, WO 2014/026997, WO2014/095775, WO 2014/095774, WO 2014/048945, WO 2014/128070; and WO2014/128111. Preferred bromodomain inhibitors preferentially orspecifically inhibit BRD4 activity. Additional

BRD4 inhibitors of use are described in, e.g., Schulze, et al., J BiomolScreen. 2014 Sep 29. PMID: 2526656; Philpott, et al., EpigeneticsChromatin. 2014 Jul. 13; 7:14; Wang, et al., Zhonghua Xue Ye Xue Za Zhi.2014 June; 35(6):528-32; and Muvva, et al, Mol Biosyst. 2014 Jul. 29;10(9):2384-97.

b. Inhibitory Nucleic Acids

Concurrently or simultaneously decreasing or inhibiting CDK9 geneexpression and BRD4 gene expression can be achieved using any method inthe art, including through the use of inhibitory nucleic acids (e.g.,small interfering RNA (siRNA), micro RNA (miRNA), antisense RNA,ribozymes, etc.). Inhibitory nucleic acids can be single-strandednucleic acids that can specifically bind to a complementary nucleic acidsequence. By binding to the appropriate target sequence, an RNA-RNA, aDNA-DNA, or an RNA-DNA duplex or triplex is formed. Such inhibitorynucleic acids can be in either the “sense” or “antisense” orientation.See, for example, Tafech, et al., Curr Med Chem (2006) 13:863-81;Mahato, et al., Expert Opin Drug Deliv (2005) 2:3-28; Scanlon, CurrPharm Biotechnol (2004) 5:415-20; and Scherer and Rossi, Nat Biotechnol(2003) 21:1457-65.

In one embodiment, the inhibitory nucleic acid can specifically bind toa target nucleic acid sequence or subsequence that encodes CDK9, BRD4 orboth CDK9 and BRD4. Administration of such inhibitory nucleic acids candecrease or inhibit the activity of CDK9 and BRD4, and consequently,cartilage degradation. Nucleotide sequences encoding CDK9 are known forseveral mammalian species, including human, e.g., NM_001261.3.Nucleotide sequences encoding BRD4 are known for several mammalianspecies, including human, e.g., NM_014299.2→NP_055114.1 short isoform;and NM_058243.2→NP_490597.1 long isoform. From known CDK9 and BRD4nucleotide sequences, one can derive a suitable inhibitory nucleic acid.

1. Antisense Oligonucleotides

In some embodiments, the inhibitory nucleic acid is an antisensemolecule. Antisense oligonucleotides are relatively short nucleic acidsthat are complementary (or antisense) to the coding strand (sensestrand) of the mRNA encoding CDK9 and/or BRD4. Although antisenseoligonucleotides are typically RNA based, they can also be DNA based.

Additionally, antisense oligonucleotides are often modified to increasetheir stability.

Without being bound by theory, the binding of these relatively shortoligonucleotides to the mRNA is believed to induce stretches of doublestranded RNA that trigger degradation of the messages by endogenousRNAses. Additionally, sometimes the oligonucleotides are specificallydesigned to bind near the promoter of the message, and under thesecircumstances, the antisense oligonucleotides may additionally interferewith translation of the message. Regardless of the specific mechanism bywhich antisense oligonucleotides function, their administration to acell or tissue allows the degradation of the mRNA encoding CDK9 and/orBRD4. Accordingly, antisense oligonucleotides decrease the expressionand/or activity of CDK9 and/or BRD4.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g.,PCT Publication No. WO 89/10134), hybridization-triggered cleavageagents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) orintercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). Tothis end, the oligonucleotide can be conjugated to another molecule.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetyl cytosine, 5-(carboxyhydroxytriethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomet-hyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine,

N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl cytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

The antisense oligonucleotide can also contain a neutral peptide-likebackbone. Such molecules are termed peptide nucleic acid (PNA)-oligomersand are described, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl.Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993) Nature 365:566.One advantage of PNA oligomers is their capability to bind tocomplementary DNA essentially independently from the ionic strength ofthe medium due to the neutral backbone of the DNA. In yet anotherembodiment, the antisense oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet a further embodiment, the antisense oligonucleotide is an-anomeric oligonucleotide. An anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides may be synthesized by standard methods known in theart, e.g., by use of an automated DNA synthesizer (such as arecommercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

The selection of an appropriate oligonucleotide can be readily performedby one of skill in the art. Given the nucleic acid sequence encodingCDK9 and/or BRD4, one of skill in the art can design antisenseoligonucleotides that bind to a target nucleic acid sequence and testthese oligonucleotides in an in vitro or in vivo system to confirm thatthey bind to and mediate the degradation of the mRNA encoding the CDK9.To design an antisense oligonucleotide that specifically binds to andmediates the degradation of a CDK9 and/or BRD4 encoding nucleic acid, itis preferred that the sequence recognized by the oligonucleotide isunique or substantially unique to the CDK9 and/or BRD4 to be inhibited.For example, sequences that are frequently repeated across an encodingsequence may not be an ideal choice for the design of an oligonucleotidethat specifically recognizes and degrades a particular message. One ofskill in the art can design an oligonucleotide, and compare the sequenceof that oligonucleotide to nucleic acid sequences that are deposited inpublicly available databases to confirm that the sequence is specific orsubstantially specific for CDK9 and/or BRD4.

A number of methods have been developed for delivering antisense DNA orRNA to cells; e.g., antisense molecules can be injected directly intothe tissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systematically.

However, it may be difficult to achieve intracellular concentrations ofthe antisense sufficient to suppress translation on endogenous mRNAs incertain instances. Therefore another approach utilizes a recombinant DNAconstruct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. For example, a vectorcan be introduced in vivo such that it is taken up by a cell and directsthe transcription of an antisense RNA. Such a vector can remain episomalor become chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al, 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct that can be introduced directly into the tissue site.Alternatively, viral vectors can be used which selectively infect thedesired tissue, in which case administration may be accomplished byanother route (e.g., systematically).

2. Small Interfering RNA (siRNA or RNAi)

In some embodiments, the inhibitory nucleic acid is a small interferingRNA (siRNA or RNAi) molecule. RNAi constructs comprise double strandedRNA that can specifically block expression of a target gene. “RNAinterference” or “RNAi” is a term initially applied to a phenomenonwhere double-stranded RNA (dsRNA) blocks gene expression in a specificand post-transcriptional manner. RNAi provides a useful method ofinhibiting gene expression in vitro or in vivo. RNAi constructs caninclude small interfering

RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleavedin vivo to form siRNAs. RNAi constructs herein also include expressionvectors (“RNAi expression vectors”) capable of giving rise totranscripts which form dsRNAs or hairpin RNAs in cells, and/ortranscripts which can produce siRNAs in vivo.

RNAi expression vectors express (transcribe) RNA which produces siRNAmoieties in the cell in which the construct is expressed. Such vectorsinclude a transcriptional unit comprising an assembly of (1) geneticelement(s) having a regulatory role in gene expression, for example,promoters, operators, or enhancers, operatively linked to (2) a “coding”sequence which is transcribed to produce a double-stranded RNA (two RNAmoieties that anneal in the cell to form an siRNA, or a single hairpinRNA which can be processed to an siRNA), and (3) appropriatetranscription initiation and termination sequences. The choice ofpromoter and other regulatory elements generally varies according to theintended host cell.

The RNAi constructs contain a nucleotide sequence that hybridizes underphysiologic conditions of the cell to the nucleotide sequence of atleast a portion of the mRNA transcript for the gene to be inhibited(i.e., a nucleic acid sequence encoding CDK9 and/or BRD4). Thedouble-stranded RNA need only be sufficiently similar to natural RNAthat it has the ability to mediate RNAi. Thus, the invention has theadvantage of being able to tolerate sequence variations that might beexpected due to genetic mutation, strain polymorphism or evolutionarydivergence. The number of tolerated nucleotide mismatches between thetarget sequence and the RNAi construct sequence is no more than 1 in 5basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50basepairs. Mismatches in the center of the siRNA duplex are mostcritical and may essentially abolish cleavage of the target RNA. Incontrast, nucleotides at the 3′ end of the siRNA strand that iscomplementary to the target RNA do not significantly contribute tospecificity of the target recognition.

Sequence identity can be optimized by sequence comparison and alignmentalgorithms known in the art (see Gribskov and Devereux, SequenceAnalysis Primer, Stockton Press, 1991, and references cited therein) andcalculating the percent difference between the nucleotide sequences by,for example, the Smith-Waterman algorithm as implemented in the BESTFITsoftware program using default parameters (e.g., University of WisconsinGenetic Computing Group). Greater than 90% sequence identity, forexample, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity,between the inhibitory RNA and the portion of the target gene ispreferred. Alternatively, the duplex region of the RNA may be definedfunctionally as a nucleotide sequence that is capable of hybridizingwith a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mMPIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours;followed by washing).

Production of RNAi constructs can be carried out by chemical syntheticmethods or by recombinant nucleic acid techniques. Endogenous RNApolymerase of the treated cell may mediate transcription in vivo, orcloned RNA polymerase can be used for transcription in vitro. The RNAiconstructs may include modifications to either the phosphate-sugarbackbone or the nucleoside, e.g., to reduce susceptibility to cellularnucleases, improve bioavailability, improve formulation characteristics,and/or change other pharmacokinetic properties. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of an nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored to allow specific genetic inhibition whileavoiding a general response to dsRNA. Likewise, bases may be modified toblock the activity of adenosine deaminase. The RNAi construct may beproduced enzymatically or by partial/total organic synthesis, anymodified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis.

Methods of chemically modifying RNA molecules can be adapted formodifying RNAi constructs (see, for example, Heidenreich et al. (1997)Nucleic Acids Res, 25:776-780; Wilson et al. (1994) J Mol Recog 7:89-98;Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al.(1997) Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate,the backbone of an RNAi construct can be modified withphosphorothioates, phosphoramidate, phosphodithioates, chimericmethylphosphonate-phosphodie-sters, peptide nucleic acids,5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g.,2′-substituted ribonucleosides, a-configuration).

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition, while lower doses may also be useful for specificapplications. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition.

In certain embodiments, the subject RNAi constructs are “smallinterfering RNAs” or “siRNAs.” These nucleic acids are around 19-30nucleotides in length, and even more preferably 21-23 nucleotides inlength, e.g., corresponding in length to the fragments generated bynuclease “dicing” of longer double-stranded RNAs. The siRNAs areunderstood to recruit nuclease complexes and guide the complexes to thetarget mRNA by pairing to the specific sequences. As a result, thetarget mRNA is degraded by the nucleases in the protein complex. In aparticular embodiment, the 21-23 nucleotides siRNA molecules comprise a3′ hydroxyl group.

The siRNA molecules of the present invention can be obtained using anumber of techniques known to those of skill in the art. For example,the siRNA can be chemically synthesized or recombinantly produced usingmethods known in the art. For example, short sense and antisense RNAoligomers can be synthesized and annealed to form double-stranded RNAstructures with 2-nucleotide overhangs at each end (Caplen, et al.(2001) Proc Natl Acad Sci USA, 98:9742-9747; Elbashir, et al. (2001)EMBO J, 20:6877-88). These double-stranded siRNA structures can then bedirectly introduced to cells, either by passive uptake or a deliverysystem of choice, such as described below.

In certain embodiments, the siRNA constructs can be generated byprocessing of longer double-stranded RNAs, for example, in the presenceof the enzyme dicer. In one embodiment, the Drosophila in vitro systemis used. In this embodiment, dsRNA is combined with a soluble extractderived from Drosophila embryo, thereby producing a combination. Thecombination is maintained under conditions in which the dsRNA isprocessed to RNA molecules of about 21 to about 23 nucleotides.

The siRNA molecules can be purified using a number of techniques knownto those of skill in the art. For example, gel electrophoresis can beused to purify siRNAs. Alternatively, non-denaturing methods, such asnon-denaturing column chromatography, can be used to purify the siRNA.In addition, chromatography (e.g., size exclusion chromatography),glycerol gradient centrifugation, affinity purification with antibodycan be used to purify siRNAs.

In certain preferred embodiments, at least one strand of the siRNAmolecules has a 3′ overhang from about 1 to about 6 nucleotides inlength, though may be from 2 to 4 nucleotides in length. Morepreferably, the 3′ overhangs are 1-3 nucleotides in length. In certainembodiments, one strand having a 3′ overhang and the other strand beingblunt-ended or also having an overhang. The length of the overhangs maybe the same or different for each strand. In order to further enhancethe stability of the siRNA, the 3′ overhangs can be stabilized againstdegradation. In one embodiment, the RNA is stabilized by includingpurine nucleotides, such as adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine nucleotide 3′ overhangs by2′-deoxythyinidine is tolerated and does not affect the efficiency ofRNAi. The absence of a 2′ hydroxyl significantly enhances the nucleaseresistance of the overhang in tissue culture medium and may bebeneficial in vivo.

In other embodiments, the RNAi construct is in the form of a longdouble-stranded RNA. In certain embodiments, the RNAi construct is atleast 25, 50, 100, 200, 300 or 400 bases. In certain embodiments, theRNAi construct is 400-800 bases in length. The double-stranded RNAs aredigested intracellularly, e.g., to produce siRNA sequences in the cell.However, use of long double-stranded RNAs in vivo is not alwayspractical, presumably because of deleterious effects which may be causedby the sequence-independent dsRNA response. In such embodiments, the useof local delivery systems and/or agents which reduce the effects ofinterferon are preferred.

In certain embodiments, the RNAi construct is in the form of a hairpinstructure (named as hairpin RNA). The hairpin RNAs can be synthesizedexogenously or can be formed by transcribing from RNA polymerase IIIpromoters in vivo. Examples of making and using such hairpin RNAs forgene silencing in mammalian cells are described in, for example,Paddison et al., Genes Dev, 2002, 16:948-58; McCaffrey et al., Nature,2002, 418:38-9; McManus et al., RNA, 2002, 8:842-50; Yu et al., ProcNatl Acad Sci USA, 2002, 99:6047-52). Preferably, such hairpin RNAs areengineered in cells or in an animal to ensure continuous and stablesuppression of a desired gene. It is known in the art that siRNAs can beproduced by processing a hairpin RNA in the cell.

In yet other embodiments, a plasmid is used to deliver thedouble-stranded RNA, e.g., as a transcriptional product. In suchembodiments, the plasmid is designed to include a “coding sequence” foreach of the sense and antisense strands of the RNAi construct. Thecoding sequences can be the same sequence, e.g., flanked by invertedpromoters, or can be two separate sequences each under transcriptionalcontrol of separate promoters. After the coding sequence is transcribed,the complementary RNA transcripts base-pair to form the double-strandedRNA.

PCT application WO 01/77350 describes an exemplary vector forbi-directional transcription of a transgene to yield both sense andantisense RNA transcripts of the same transgene in a eukaryotic cell.Accordingly, in certain embodiments, the present invention provides arecombinant vector having the following unique characteristics: itcomprises a viral replicon having two overlapping transcription unitsarranged in an opposing orientation and flanking a transgene for an RNAiconstruct of interest, wherein the two overlapping transcription unitsyield both sense and antisense RNA transcripts from the same transgenefragment in a host cell.

RNAi constructs can comprise either long stretches of double strandedRNA identical or substantially identical to the target nucleic acidsequence or short stretches of double stranded RNA identical tosubstantially identical to only a region of the target nucleic acidsequence. Exemplary methods of making and delivering either long orshort RNAi constructs can be found, for example, in WO 01/68836 and WO01/75164.

Exemplary RNAi constructs that specifically recognize a particular gene,or a particular family of genes can be selected using methodologyoutlined in detail above with respect to the selection of antisenseoligonucleotide. Similarly, methods of delivery RNAi constructs includethe methods for delivery antisense oligonucleotides outlined in detailabove.

3. Ribozymes

In some embodiments, the inhibitory nucleic acid is a ribozyme.Ribozymes molecules designed to catalytically cleave an mRNA transcriptscan also be used to prevent translation of mRNA (See, e.g., PCTInternational Publication WO 90/11364; Sarver et al., 1990, Science247:1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleavemRNA at site-specific recognition sequences can be used to destroyparticular mRNAs, the use of hammerhead ribozymes is preferred.Hammerhead ribozymes cleave mRNAs at locations dictated by flankingregions that form complementary base pairs with the target mRNA. Thesole requirement is that the target mRNA have the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully inHaseloff and Gerlach, 1988, Nature, 334:585-591.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; WO 88/04300; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site that hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes that targeteight base-pair active site sequences.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) and canbe delivered to cells in vitro or in vivo. A preferred method ofdelivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive pol III or pol II promoter, so thattransfected cells will produce sufficient quantities of the ribozyme todestroy targeted messages and inhibit translation. Because ribozymesunlike antisense molecules, are catalytic, a lower intracellularconcentration is required for efficiency.

DNA enzymes incorporate some of the mechanistic features of bothantisense and ribozyme technologies. DNA enzymes are designed so thatthey recognize a particular target nucleic acid sequence, much like anantisense oligonucleotide, however much like a ribozyme they arecatalytic and specifically cleave the target nucleic acid.

There are currently two basic types of DNA enzymes, and both of thesewere identified by Santoro and Joyce (see, for example, U.S. Pat. No.6,110,462). The 10-23 DNA enzyme comprises a loop structure whichconnect two arms. The two arms provide specificity by recognizing theparticular target nucleic acid sequence while the loop structureprovides catalytic function under physiological conditions.

Briefly, to design an ideal DNA enzyme that specifically recognizes andcleaves a target nucleic acid, one of skill in the art must firstidentify the unique target sequence. This can be done using the sameapproach as outlined for antisense oligonucleotides. Preferably, theunique or substantially sequence is a G/C rich of approximately 18 to 22nucleotides. High G/C content helps insure a stronger interactionbetween the DNA enzyme and the target sequence.

When synthesizing the DNA enzyme, the specific antisense recognitionsequence that will target the enzyme to the message is divided so thatit comprises the two arms of the DNA enzyme, and the DNA enzyme loop isplaced between the two specific arms.

Methods of making and administering DNA enzymes can be found, forexample, in U.S. Pat. No. 6,110,462. Similarly, methods of delivery DNAribozymes in vitro or in vivo include methods of delivery RNA ribozyme,as outlined in detail above. Additionally, one of skill in the art willrecognize that, like antisense oligonucleotide, DNA enzymes can beoptionally modified to improve stability and improve resistance todegradation.

4. Formulation and Administration

In therapeutic applications, a combination of the one or more CDK9inhibitors and the one or more BRD4 inhibitors can be administered to anindividual who has suffered a traumatic injury to cartilage tissue, whohas undergone surgery to repair cartilage tissue and/or who has receiveda cartilage allograft. Compositions that contain a combination of one ormore CDK9 inhibitors and one or more BRD4 inhibitors are administered toa patient in an amount sufficient to suppress the undesirableinflammation and to eliminate or at least partially arrest symptomsand/or complications. An amount adequate to accomplish this is definedas a “therapeutically effective dose.” Amounts effective for this usewill depend on, e.g., the inhibitor composition, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician. A combination of inhibitors of CDK9 and BRD4activity can be administered chronically or acutely to reduce, inhibitor prevent cartilage degradation and post traumatic osteoarthritis. Incertain instances, it will be appropriate to administer one or moreinhibitors of CDK9 activity and one or more inhibitors of BRD4 activityprophylactically, for instance in subjects at risk of or suspected ofdeveloping cartilage degradation and/or post traumatic osteoarthritis.

Alternatively, DNA or RNA that inhibits expression of one or moresequences encoding a CDK9 protein, such as an antisense nucleic acid, asmall-interfering nucleic acid (i.e., siRNA), a micro RNA (miRNA), or anucleic acid that encodes a peptide that blocks expression or activityof a CDK9 can be introduced into patients to achieve inhibition. U.S.Pat. No. 5,580,859 describes the use of injection of naked nucleic acidsinto cells to obtain expression of the genes which the nucleic acidsencode.

Therapeutically effective amounts of CDK9 inhibitor or enhancercompositions of the present invention generally range for the initialadministration (that is for therapeutic or prophylactic administration)from about 0.1 μg to about 10 mg, or less, of CDK9 inhibitor and/or BRD4inhibitor for a 70 kg patient, usually from about 1.0 μg to about 1 mg,or less, for example, between about 10 μg to about 0.1 mg (100 μg), orless, particularly when the CDK9 inhibitor and BRD4 inhibitor areco-administered. Whereas the combination of the CDK9 inhibitor and BRD4inhibitor are together efficacious, the amounts of the individual CDK9inhibitor and/or BRD4 inhibitor can be non-efficacious. Typically, lowerdoses are initially administered and incrementally increased until adesired efficacious dose is reached. These doses can be followed byrepeated administrations over weeks to months depending upon thepatient's response and condition by evaluating symptoms associated withcartilage degradation and/or post-traumatic osteoarthritis.

For prophylactic use, administration should be given to subjects at riskfor or suspected of developing cartilage degradation and/orpost-traumatic osteoarthritis. Therapeutic administration may beginconcurrently with surgical and/or allograft procedures, and/or as soonas possible after traumatic injury or surgery. This is often followed byrepeated administration until at least symptoms are substantially abatedand for a period thereafter. In some embodiments, the inhibitor of CDK9and the inhibitor of BRD4 are co-administered within 10 days, e.g.,within 9, 8, 7, 6, 5, 4, 3, 2, 1 days, e.g., within 24, 20, 18, 16, 14,12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours after experiencing traumaticinjury. In some embodiments, the subject has undergone surgery to repairdamaged cartilage tissue. In some embodiments, the subject has receivedan osteochondral explant, e.g., a cartilage allograft. In someembodiments, the inhibitor of CDK9 and the inhibitor of BRD4 areco-administered concurrently with or prior to surgery. In someembodiments, the inhibitor of CDK9 and the inhibitor of BRD4 areco-administered within 10 days, e.g., within 9, 8, 7, 6, 5, 4, 3, 2, 1days, e.g., within 24, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1hours after surgery. In some embodiments, the inhibitor of CDK9 and theinhibitor of BRD4 are co-administered over a course of 10 days, e.g.,over 9, 8, 7, 6, 5, 4, 3, 2, 1 days. In various embodiments, theinhibitor of CDK9 and the inhibitor of BRD4 are co-administered every 2days, every day, or twice daily, as appropriate.

The combination of one or more CDK9 inhibitors and one or more BRD4inhibitors for therapeutic or prophylactic treatment are intended forsystemic (e.g., parenteral, topical, oral, transdermal) or local (e.g.,intralesional) administration. Preferably, the compositions areformulated for oral administration. In certain embodiments, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, intranasally, inhalationally, subcutaneously,intradermally, or intramuscularly. Compositions are also suitable fororal administration. Thus, the invention provides compositions forparenteral administration which comprise a solution of the CDK9inhibiting agent and the BRD4 inhibiting agent dissolved or suspended inan acceptable carrier, preferably an aqueous carrier. A variety ofaqueous carriers may be used, e.g., water, buffered water, 0.9% saline,0.3% glycine or another suitable amino acid, hyaluronic acid and thelike. These compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

In embodiments where one or both of the CDK9 inhibitor and the BRD4inhibitor are small organic compounds, the compound (e.g., flavopiridol,JQ1, GSK525762A) and/or an analog thereof can be administered orally,parenterally, (intravenously (IV), intramuscularly (IM), depo-IM,subcutaneously (SQ), and depo-SQ), sublingually, intranasally(inhalation), intrathecally, transdermally (e.g., via transdermalpatch), topically, ionophoretically or rectally. Typically the dosageform is selected to facilitate delivery to the brain (e.g., passagethrough the blood brain barrier). In this context it is noted that thecompounds described herein are readily delivered to the brain. Dosageforms known to those of skill in the art are suitable for delivery ofthe compound.

In varying embodiments, the CDK9 inhibitor and the BRD4 inhibitor areco-administered intravenously. In embodiments where the CDK9 inhibitoris flavopiridol, dosing can be in accordance with concentrations andscheduling reported in the art. For example, in various embodiments,flavopiridol is administered intravenously in a concentration range ofabout 10 to about 105 mg/m² in infusions delivered over 1 to 4 hours, asappropriate. See, e.g., Ramaswamy, et al,. Invest New Drugs. (2012)30(2):629-38; Phelps, et al., Blood. (2009) 113(12):2637-45; and Byrd,et al., Blood. (2007) 109(2):399-404. In varying embodiments, JQ1 can beadministered at nanomolar concentrations, e.g., 10-5000 nM, e.g.,50-1000 nM, e.g., 50-500 nM. In varying embodiments, JQ1 is administeredat a dose of about 50 mg/kg. See, e.g., Trabucco, et al., Clin CancerRes. 2014 Jul 9, PMID 25009295; and Spiltoir, et al., J Mol CellCardiol. 2013 October; 63:175-9.

Compositions are provided that contain therapeutically effective amountsof the compound. The compounds are preferably formulated into suitablepharmaceutical preparations such as tablets, capsules, or elixirs fororal administration or in sterile solutions or suspensions forparenteral administration. Typically the compounds described above areformulated into pharmaceutical compositions using techniques andprocedures well known in the art.

These active agents (e.g.,flavopiridol and/or analogs thereof) can beadministered in the “native” form or, if desired, in the form of salts,esters, amides, prodrugs, derivatives, and the like, provided the salt,ester, amide, prodrug or derivative is suitable pharmacologicallyeffective, e.g., effective in the present method(s). Salts, esters,amides, prodrugs and other derivatives of the active agents can beprepared using standard procedures known to those skilled in the art ofsynthetic organic chemistry and described, for example, by March (1992)Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed.N.Y. Wiley-Interscience.

Methods of formulating such derivatives are known to those of skill inthe art. For example, the disulfide salts of a number of delivery agentsare described in PCT Publication WO 2000/059863 which is incorporatedherein by reference. Similarly, acid salts of therapeutic peptides,peptoids, or other mimetics, and can be prepared from the free baseusing conventional methodology that typically involves reaction with asuitable acid.

Generally, the base form of the drug is dissolved in a polar organicsolvent such as methanol or ethanol and the acid is added thereto. Theresulting salt either precipitates or can be brought out of solution byaddition of a less polar solvent. Suitable acids for preparing acidaddition salts include, but are not limited to both organic acids, e.g.,acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, orotic acid, and the like, as well as inorganic acids,e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt can be reconvertedto the free base by treatment with a suitable base. Certain particularlypreferred acid addition salts of the active agents herein include halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of the active agents of thisinvention are prepared in a similar manner using a pharmaceuticallyacceptable base such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, calcium hydroxide, trimethylamine, or the like. In certainembodiments basic salts include alkali metal salts, e.g., the sodiumsalt, and copper salts.

For the preparation of salt forms of basic drugs, the pKa of thecounterion is preferably at least about 2 pH lower than the pKa of thedrug. Similarly, for the preparation of salt forms of acidic drugs, thepKa of the counterion is preferably at least about 2 pH higher than thepKa of the drug. This permits the counterion to bring the solution's pHto a level lower than the pHmax to reach the salt plateau, at which thesolubility of salt prevails over the solubility of free acid or base.The generalized rule of difference in pKa units of the ionizable groupin the active pharmaceutical ingredient (API) and in the acid or base ismeant to make the proton transfer energetically favorable. When the pKaof the API and counterion are not significantly different, a solidcomplex may form but may rapidly disproportionate (e.g., break down intothe individual entities of drug and counterion) in an aqueousenvironment.

Preferably, the counterion is a pharmaceutically acceptable counterion.Suitable anionic salt forms include, but are not limited to acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

In various embodiments preparation of esters typically involvesfunctionalization of hydroxyl and/or carboxyl groups that are presentwithin the molecular structure of the active agent. In certainembodiments, the esters are typically acyl-substituted derivatives offree alcohol groups, e.g., moieties that are derived from carboxylicacids of the formula RCOOH where R is alky, and preferably is loweralkyl. Esters can be reconverted to the free acids, if desired, by usingconventional hydrogenolysis or hydrolysis procedures.

Amides can also be prepared using techniques known to those skilled inthe art or described in the pertinent literature. For example, amidesmay be prepared from esters, using suitable amine reactants, or they maybe prepared from an anhydride or an acid chloride by reaction withammonia or a lower alkyl amine.

The concentration of CDK9 inhibiting agents and the BRD4 inhibitingagents in the pharmaceutical formulations can vary widely, i.e., fromless than about 0.1%, usually at or at least about 2% to as much as 20%to 50% or more by weight, and will be selected primarily by fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

The CDK9 inhibitors and/or the BRD4 inhibitors may also be administeredvia liposomes, which can be designed to target the conjugates to aparticular tissue, for example, cartilage tissue. Liposomes includeemulsions, foams, micelles, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. In thesepreparations, the peptide, nucleic acid or organic compound to bedelivered is incorporated as part of a liposome, alone or in conjunctionwith a molecule which binds to, e.g., a receptor prevalent among thedesired cells, or with other therapeutic compositions. Thus, liposomesfilled with a desired peptide, nucleic acid, small molecule or conjugatecan be directed to the damaged or injured lesion, for example, cartilagetissue, joints, injured lesions, where the liposomes then deliver theselected CDK9 inhibitor and/or BRD4 inhibitor compositions. Liposomesfor use in the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidliability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467(1980), U.S. Pat. Nos.4,235,871, 4,501,728 and 4,837,028.

The targeting of liposomes using a variety of targeting agents is wellknown in the art (see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044).For targeting to desired cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the target cells. A liposome suspensioncontaining a CDK9 inhibitor and/or BRD4 inhibitor may be administeredintravenously, locally (e.g., intralesionally), topically, etc., in adose which varies according to, inter alia, the manner ofadministration, the conjugate being delivered, and the stage of thedisease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more conjugates, and more preferably at aconcentration of 25%-75%.

For aerosol administration, the inhibitors are preferably supplied in asuitable form along with a surfactant and propellant. Typicalpercentages of CDK9 inhibitors and BRD4 inhibitors are 0.01%-20% byweight, preferably 1%-10%. The surfactant must, of course, be nontoxic,and preferably soluble in the propellant. Representative of such agentsare the esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides may be employed. The surfactant may constitute0.1%-20% by weight of the composition, preferably 0.25-5%. The balanceof the composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

An effective treatment is indicated by a decrease in observed symptoms(e.g., pain, swelling, joint mobility) as measured according to aclinician or reported by the patient. Alternatively, methods fordetecting levels of specific CDK9 activities and/or BRD4 activities canbe used. Standard assays for detecting CDK9 activity and/or BRD4activity are described herein. Again, an effective treatment isindicated by a substantial reduction in activity of CDK9 and/or BRD4. Asused herein, a “substantial reduction” in CDK9 activity and/or BRD4activity refers to a reduction of at least about 30% in the test samplecompared to an untreated control. Preferably, the reduction is at leastabout 50%, more preferably at least about 75%, and most preferably CDK9and/or BRD4 activity levels are reduced by at least about 90% in asample from a treated mammal compared to an untreated control. In someembodiments, the CDK9 activity and/or BRD4 activity is completelyinhibited.

5. Matrices Comprising an Inhibitor of CDK9 and an Inhibitor of BRD4

In various embodiments, a combination of one or more CDK9 inhibitors andone or more BRD4 inhibitors can be contained within a matrix or a depot.The matrix can serve, in one capacity, as a delivery vehicle for thecomposition to be delivered to the site of a cartilage lesion or a bonelesion. The matrix also provides a suitable scaffold upon whichcartilage repair and regeneration can occur. In one embodiment, thematrix is bioresorbable or biodegradable.

In various embodiments, the matrix can be formed of any material that issuitable for in vivo use, and which provides the characteristicsfacilitating cartilage repair or bone repair in the presence of aninhibitor of CDK9 and an inhibitor of BRD4. The matrix can be formed ofmaterials which include, but are not limited to, synthetic polymersand/or a ground substance. Preferred ground substances include naturalpolymers and proteoglycans. Natural polymers include, but are notlimited to collagen, elastin, reticulin and analogs thereofProteoglycans include, but are not limited to, anyglycosaminoglycan-containing molecules. Particularly preferredglycosaminoglycans include chondroitin sulfate, dermatan sulphate,heparan sulphate, keratan sulphate and hyaluronan. Other preferredground substances include, but are not limited to, type I collagen, typeII collagen, type III collagen, type IV collagen and hyaluronic acid.Preferred synthetic polymers include poly(lactic acid) and poly(glycolicacid).

In one embodiment of the present invention, the matrix includescollagen. For example, the matrix can contain from about 20% to about100% collagen by dry weight of the matrix, for example, from about 50%to about 100% collagen by dry weight of the matrix, for example, fromabout 75% to about 100% collagen by dry weight of the matrix.

A matrix suitable for use with one or more inhibitors of CDK9 and one ormore inhibitors of BRD4 can include materials in any suitable form foruse in repairing a cartilage lesion or a bone lesion, including asponge, a membrane, a film or a gel. In one embodiment, a suitablerepair matrix includes demineralized bone matrix, synthetic bone graftsubstitute, autograft tissue, allograft tissue and/or xenograft tissue.In some embodiments, the matrix is formulated for use as a bone graft,for example, as a spinal graft.

Suitable methods for associating an inhibitor of CDK9 and an inhibitorof BRD4 with a matrix include any method which allows the inhibitors tobe delivered to a site of cartilage repair or bone repair together withthe matrix such that the cartilage repair or bone repair product iseffective to repair and/or regenerate cartilage or bone at the site.Such methods of association include, but are not limited to, suspensionof the composition within the matrix, freeze-drying of the compositiononto a surface of the matrix and suspension within the matrix of acarrier/delivery formulation containing the composition. Additionally,the one or more inhibitors of CDK9 and the one or more inhibitors ofBRD4 can be associated with the matrix prior to placement of the productinto a cartilage lesion (i.e., the association of the composition withmatrix occurs ex vivo) or alternatively, the matrix can first beimplanted into a lesion, followed by association of the one or moreinhibitors of CDK9 and the one or more inhibitors of BRD4 with thematrix, such as by injection into or on top of the matrix (i.e., theassociation of the composition with matrix occurs in vivo).

The combination of one or more inhibitors of CDK9 and one of moreinhibitors of BRD4 can contain additional delivery formulations orcarriers which enhance the association of the composition with thematrix, which enhance the delivery of the composition to the appropriatecells and tissue at the site of the lesion, and which assist incontrolling the release of the factors in the composition at the site ofthe lesion. Suitable delivery formulations include carriers, which, asused herein, include compounds that increase the half-life of acartilage-inducing composition in the treated animal. Suitable carriersinclude, but are not limited to, polymeric controlled release vehicles,biodegradable implants, liposomes, bacteria, viruses, oils, cells,esters, and glycols. Preferably, the matrices are bioresorbable orbiodegradable.

The combination of one or more inhibitors of CDK9 and one or moreinhibitors of BRD4 are present in the matrix at a concentration that iseffective to induce, at the site of a cartilage lesion or a bone lesion,one or more of: cellular infiltration, cellular proliferation,angiogenesis, and cellular differentiation to type II collagen-producingchondrocytes. Individually, one or more inhibitors of CDK9 and/or one ormore inhibitors of BRD4 can be present at non-efficaciousconcentrations. In varying embodiments, the one or more inhibitors ofCDK9 and one or more BRD4 inhibitors are present in the matrices at aconcentration that the combination is effective to induce cartilagerepair and/or regeneration at the site of a cartilage lesion or a bonelesion. One of skill in the art will be able to adjust the concentrationof proteins and/or nucleic acid molecules in the composition dependingon the types and number of proteins to be provided by the composition,and the delivery vehicle used.

The matrices can also contain one or more substances that non-covalentlyattach to the one or more inhibitors of CDK9 and the one or moreinhibitors of BRD4 in the composition and thus, modify the release rateof the growth factor. Such substances include, but are not limited to,any ground substance or other polymeric substance. As used herein, aground substance is defined as the non-living matrix of connectivetissue, which includes natural polymers and proteoglycans. Naturalpolymers include, but are not limited to collagen, elastin, reticulinand analogs thereof. Proteoglycans include, but are not limited to anyglycosaminoglycan-containing molecules, and include chondroitin sulfate,dermatan sulphate, heparan sulphate, keratan sulphate and hyaluronan.Preferred ground substances include, but are not limited to, type Icollagen, type II collagen, type III collagen, type IV collagen andhyaluronic acid. Preferred other polymeric substances include, but arenot limited to, poly(lactic acid) and poly(glycolic acid).

In a further embodiment, the matrices can include one or more types ofcells which are provided to further enhance chondrogenesis at the siteof the cartilage lesion. Such cells include, but are not limited to,fibrochondrocytes, chondrocytes, mesenchymal precursors, and any othercell that can serve as a chondrocyte precursor. Such cells can beassociated with the composition and the matrix by any of the methodsdescribed above.

In some aspects of the present invention, matrices comprising the one ormore inhibitors of CDK9 and one or more inhibitors of BRD4 furthercomprise at least one bone matrix protein. As used herein, “bone matrixproteins” are any of a group of proteins known in the art to be acomponent of or associated with the minute collagenous fibers and groundsubstances which form bone matrix. In various embodiments, the matricescomprise a bone matrix protein that is a member of the TGF-βsuperfamily, a growth factor protein and/or Cartilage Oligomeric MatrixProtein (COMP). Bone matrix proteins can also include, but are notlimited to, osteocalcin, osteonectin, bone sialoprotein (BSP),lysyloxidase, cathepsin L pre, osteopontin, matrix GLA protein (MGP),biglycan, decorin, proteoglycan-chondroitin sulfate III (PG-CS III),bone acidic glycoprotein (BAG-75), thrombospondin (TSP) and/orfibronectin. Preferably, bone matrix proteins suitable for use with theproduct of the present invention include one or more of: osteocalcin,osteonectin, MGP, TSP, BSP, lysyloxidase and cathepsin L pre. In oneembodiment, the at least one bone matrix protein includes at leastosteocalcin, osteonectin, BSP, lysyloxidase and cathepsin L pre. Aparticularly preferred bone matrix protein is MGP, and more preferred isosteonectin, and most preferred is TSP.

The matrices comprising the one or more inhibitors of CDK9 and the oneor more inhibitors of BRD4 are useful for repairing a variety of defectsin cartilage, including both tears and segmental defects in bothvascular and avascular cartilage tissue. The product is particularlyuseful for repairing defects in hyaline (e.g., articular) and/orfibrocartilage (e.g., meniscal). For example, matrices comprising one ormore inhibitors of CDK9 and one or more inhibitors of find use promotingrepair of a meniscal radial tear; a meniscal triple bucket handle tear;a longitudinal tear in the avascular area of a meniscus; or a meniscalsegmental lesion.

Because cartilage defects and bone defects (i.e., lesions) can occur ina variety of shapes, sizes, and locations, a matrix comprising the oneor more inhibitors of CDK9 and the one or more inhibitors of BRD4 is ofa shape and size sufficient to conform to a specific defect in thecartilage or the bone of the patient to be treated. Preferably, thematrix, when used in the repair of a cartilage defect or bone defect,achieves a geometry at the defect site that is suitable to provide atherapeutic benefit to the patient. Such a therapeutic benefit can beany improvement in a patient's health and well-being that is related toa correction of the cartilage defect or the bone defect, and preferably,the therapeutic benefit includes the repair of the defect such that thenatural configuration of the cartilage or the bone is at least partiallyrestored. The matrix can be fixed or implanted directly into a cartilagelesion or a bone lesion.

6. Compositions and Kits

In a related aspect, the invention provides compositions comprising anosteochondral explant (e.g., ex vivo cartilage tissue) and/orchondrocytes in a solution comprising an inhibitor of cyclin-dependentkinase 9 (CDK9) and an inhibitor of bromodomain containing 4 (BRD4). Insome embodiments, the osteochondral explant is allograft cartilage. Insome embodiments, the inhibitor of CDK9 is a small organic compound,e.g., flavopiridol, or analogs and salts thereof In some embodiments,the inhibitor of BRD4 is a small organic compound, e.g., JQ1,GSK525762A, or analogs and salts thereof. In some embodiments, theosteochondral explant is submerged in the solution comprising theinhibitor of CDK9 and the inhibitor of BRD4. In varying embodiments, thesolution is an aqueous solution, e.g., a physiologically isotonicsolution. In some embodiments, the solution comprises flavopiridol at aconcentration in the range of about 100 nM to about 1000 nM, e.g., about300 nM, or less. In some embodiments, the solution comprises JQ1 and/orGSK525762A at a concentration in the range of about 100 nM to about 1000nM, e.g., about 300 nM, or less. When the solution contains acombination of a CDK9 inhibitor (e.g., flavopiridol) and a BRD4inhibitor (e.g., JQ1 and/or GSK525762A), one or both compounds can be ata non-efficacious concentration, e.g., 75%, 50% or 25% of aconcentration in the range of about 100 nM to about 1000 nM, e.g., about300nM. The solution may contain additional pharmaceutically acceptableexcipients, described herein. In some embodiments, the composition isprovided as a packaged kit.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Inhibition of CDK9 and BRD4 in Chondrocytes Under InflammatoryStimuli Materials and Methods

Treatment of Chondrocytes.

Primary chondrocytes were isolated from cartilage of healthy humandonors with IRB approval and cultured in DMEM with 10% FBS. Statisticaldesign-of-experiment was performed using JMP 10.0 software to identifythe minimum concentration of each drug alone, or in combination,required for complete inhibition of IL-1β-induced iNOS transcription.Chondrocytes were treated with either 10 ng/ml of IL-1β or 10 ng/ml ofTNF-a as inflammatory stimuli for 5 hours, and with 5 drug combinationsas follows;

-   -   1) no drug without cytokine stimulus (control group)    -   2) no drug with stimulus (stimulus group)    -   3) JQ1 with stimulus (J group)    -   4) Flavopiridol with stimulus (F group)    -   5) JQ1 and Flavopiridol with stimulus (JF group).        The cells were harvested for RNA extraction.

Quantitative Real-Time RT-PCR.

Total RNA was extracted from the chondrocytes (n=3 donors) usingmiRNeasy Mini Kit (Qiagen). After the first-strand Cdna was synthesizedusing Quantitect Reverse Transcription Kit (Qiagen), the converted cDNAsamples were amplified in triplicate by a 7900HT real-time PCR system(Applied Biosystems) with gene specific probes and normalized to 18srRNA.

Microarray Analysis.

Affymetrix GeneChip Human Gene 2.0 ST Array

Analysis was performed with total RNA extracted from the chondrocytes(n=2 donors) of 5 groups as described above. First, we selected geneswhose expression levels in the stimulus group were 5-fold higher than inthe control group, and were repressed by drugs (JQ1, Flavopiridol orboth). GeneSpring software identified genes that showed significantdifference among the 5 groups by one-way ANOVA. The differentiallyregulated genes were input into the Ingenuity Pathway Analysis Softwareto evaluate the changes in canonical pathways.

Results are shown in FIGS. 3, 4 and 7. Inhibition of CDK9 and BRD4 withFlavopiridol and JQ1 effectively repressed a panel of pro-inflammatoryand catabolic genes. The combination of JQ1 and Flavopiridol showed asynergistic interaction, with similar or better repression ofinflammatory response achieved at reduced drug doses (e.g.,sub-therapeutic or non-efficacious for the individual agents). Brd4 andCdk9 regulate the transcription of primary response inflammatory genesnot only through common mechanisms, but also through independentmechanisms.

Example 2 Transcriptional Control of Trauma-Induced SystemicInflammatory Response

Inhibition of Cdk9 after knee trauma is highly effective at preventingthe local inflammatory response, and is further effective in preventingpost-traumatic osteoarthritis (Yik, et al., Arthritis & Rheumatology(2014) 66, 1537-1546). The method of treating SIRS and traumaticsystemic inflammation is an expansion of our traumatic knee injurystudies into other orthopaedic related traumas, such as multiple longbone fractures, that are a major risk factor for systemic inflammation.Systemic inflammation significantly delays fracture healing andincreases medical costs (Bastian, et al., Journal of Leukocyte Biology(2011) 89, 669-673). Therefore, an effective treatment for SIRS wouldbenefit patients with orthopaedic trauma. As discussed below, our dataclearly demonstrate that inhibiting CDK9 alone and concurrentlyinhibiting Cdk9 and Brd4 effectively suppresses the activation ofprimary inflammatory response genes resulting from numerousimmunological challenges and prevents inflammation induced secondaryhealth effects.

Cdk9 inhibition blocks multiple upstream inflammatory signals. Todemonstrate that Cdk9 regulates the common bottle-neck of inflammatorygene activation, human primary chondrocytes were treated separately withfour different pro-inflammatory stimuli, IL-1β, lipopolysaccharide(LPS), TNF (in FIG. 5A), or IL-6 (in FIG. 5C), which activate differentcell surface receptors and signaling pathways (see FIG. 1). The mRNAinduction of the primary inflammatory gene inducible nitric oxidesynthase (iNOS) was then determined. The results showed that Cdk9inhibition by Flavopiridol markedly suppressed iNOS induction by allfour inflammatory stimuli (FIG. 5A & C). Moreover, the observed effectswere specifically mediated by Cdk9 because siRNA-mediated knocked downof Cdk9 also prevented iNOS induction by IL-1β treatment (FIG. 5B).Besides iNOS, we have shown that the four stimuli also induced mRNA ofother inflammatory cytokines and catabolic genes such as multiple matrixmetalloproteases (MMPs), but all the induction was suppressed byFlavopiridol (Yik, et al., Arthritis & Rheumatology (2014) 66,1537-1546). Finally, Cdk9 inhibition prevented IL-6 mRNA induction fromcartilage explants subjected to mechanical injuries (FIG. 6A), and theassociated apoptosis (FIG. 6B), indicating Cdk9 inhibition protectscartilage from the deleterious effects of inflammation and injuries.

In summary, blocking Cdk9 alone, and concurrently blocking Cdk9 and

Brd4, prevents the transcription of inflammatory genes in response todiverse signals including IL-1(3, LPS, TNF, IL-6, and mechanical injury.These data confirm the central role of transcriptional activation instress response, and strongly indicate that this approach would also besuccessful in blocking SIRS after severe trauma. Cdk9 inhibitioneffectively suppresses activation of most downstream inflammatoryresponse genes. We next performed microarray screening to demonstratethe effects of Cdk9 inhibition on the activation of a broad spectrum ofinflammatory response genes. Primary human chondrocytes in culture weretreated with IL-1β as an inflammatory stimulus, in the presence orabsence of Flavopiridol and/or JQ1 for 5 hours. Microarrays detected 873genes within the entire transcriptome that were induced >1.5-fold byIL-1β. Heat map representation of relative gene expression levels showedthat JQ1 and Flavopiridol preferentially suppressed a distinctpopulation of these 873 genes (FIG. 7A, compare lanes 2 and 3). Asynergistic effect was observed in co-treatment of JQ1 and Flavopiridolat about 20-25% of the original doses, and produced a gene expressionprofile that most closely resembles untreated samples (FIG. 7A, comparelanes 4 and 5). These data highlight the advantage of the synergisticand complementary effects of using both Flavopiridol and JQ1 foranti-inflammatory therapy

Our results also demonstrate the effectiveness and the magnitude of Cdk9inhibitors in suppressing the induction of primary inflammatory genes.For example, induction of nearly half of the 873 IL-1β-induced genes wassuppressed by Flavopiridol to levels that were at least 75% less thantheir maximum induction values (FIG. 7B, yellow). Only ˜5% of the 873genes were not suppressed at all by Flavopiridol (FIG. 7B, grey).Furthermore, strong synergy was seen between with Flavopiridol and JQ1.Co-treatment with the two drugs achieved better suppression even at areduced dosages of 60 nM Flavopiridol and 250 nM JQ1 (FIGS. 3 and 7B),when compared to the dosages for single treatments of 250 nMFlavopiridol or 1200 nM JQ1. Reducing the dosages will minimizepotential off-target effects. Moreover, our microarray data demonstratedthat only inducible genes were affected by Cdk9 inhibition, while theuninduced genes and housekeeping genes were not affected, indicatingthat Cdk9 inhibition, alone and concurrently with Brd4 inhibition, hasminimal off-target effects within the short duration of our experiments.Taken together, the results provided herein and our published data (Yik,et al., Arthritis & Rheumatology (2014) 66, 1537-1546) demonstrate theeffectiveness of Cdk9 inhibitors, alone and in combination with Brd4inhibitors, as anti-inflammatory agents that block most inflammatorygene transcription.

Our data herein provide strong evidence that inhibiting Cdk9, alone andin combination with Brd4 inhibition, is a viable anti-inflammatorystrategy that sets itself apart from conventional anti-inflammatoryagents for the treatment of SIRS. In vitro Cdk9 inhibition not onlyblocks multiple upstream inflammatory signals, but also suppresses theinduction of most downstream inflammatory response genes. Second, invivo Cdk9 inhibition, alone and concurrently with Brd4 inhibition,reduces tissue inflammation and catabolic responses in our mouse kneeinjury model. Most importantly, using the LPS-induced systemicinflammation mouse model, we demonstrate that Cdk9 inhibition markedlysuppresses plasma levels of the systemic inflammatory marker IL-6. Takentogether, these data strongly support that inhibiting Cdk9, alone and incombination with inhibiting Brd4, is a viable strategy to prevent andtreat SIRS resulting from severe trauma.

REFERENCES

-   1. Brandt, K. D.; Dieppe, P.; and Radin, E.: Etiopathogenesis of    osteoarthritis. The Medical clinics of North America, 93(1): 1-24,    xv, 2009.-   2. Anderson, D. D.; Chubinskaya, S.; Guilak, F.; Martin, J. A.;    Oegema, T. R.; Olson, S. A.; and Buckwalter, J. A.: Post-traumatic    osteoarthritis: Improved understanding and opportunities for early    intervention. Journal of orthopaedic research : official publication    of the Orthopaedic Research Society, 29(6): 802-9, 2011.-   3. Lewis, J. S. et al.: Acute joint pathology and synovial    inflammation is associated with increased intra-articular fracture    severity in the mouse knee. Osteoarthritis and cartilage/OARS,    Osteoarthritis Research Society, 2011.-   4. Brown, T. D.; Johnston, R. C.; Saltzman, C. L.; Marsh, J. L.; and    Buckwalter, J. A.: Posttraumatic osteoarthritis: a first estimate of    incidence, prevalence, and burden of disease. J Orthop Trauma,    20(10): 739-44, 2006.-   5. AAOS: http://orthoinfo.aaos.org/topic.cfm?topic=a00297. 2009.-   6. Bottoni, C.: Anterior Cruciate Ligament Reconstructions in    Active-Duty Military Patients. Operative Techniques in Sports    Medicine, 13(3): 169-175, 2005.-   7. Lohmander, L. S.; Englund, P. M.; Dahl, L. L.; and Roos, E. M.:    The long-term consequence of anterior cruciate ligament and meniscus    injuries: osteoarthritis. The American journal of sports medicine,    35(10): 1756-69, 2007.-   8. Firestein, G. S., and Kelley, W. N.: Kelley's textbook of    rheumatology. Edited, Philadelphia, Pa., Saunders/Elsevier, 2009.-   9. Nielsen, A. B., and Yde, J.: Epidemiology of acute knee injuries:    a prospective hospital investigation. The Journal of Trauma, 31(12):    1644-8, 1991.

10. Buckwalter, J. A., and Brown, T. D.: Joint injury, repair, andremodeling: roles in post-traumatic osteoarthritis. ClinicalOrthopaedics and Related Research, (423): 7-16, 2004.

-   11. Roos, H.; Adalberth, T.; Dahlberg, L.; and Lohmander, L. S.:    Osteoarthritis of the knee after injury to the anterior cruciate    ligament or meniscus: the influence of time and age. Osteoarthritis    and cartilage/OARS, Osteoarthritis Research Society, 3(4): 261-7,    1995.-   12. Felson, D. T.: Osteoarthritis in 2010: New takes on treatment    and prevention. Nature reviews. Rheumatology, 7(2): 75-6, 2011.-   13. Lotz, M. K.: New developments in osteoarthritis. Posttraumatic    osteoarthritis: pathogenesis and pharmacological treatment options.    Arthritis Res Ther, 12(3): 211, 2010.-   14. Catterall, J. B.; Stabler, T. V.; Flannery, C. R.; and Kraus, V.    B.: Changes in serum and synovial fluid biomarkers after acute    injury (NCT00332254). Arthritis Research & Therapy, 12(6): R229,    2010.-   15. Hargreaves, D. C.; Horng, T.; and Medzhitov, R.: Control of    inducible gene expression by signal-dependent transcriptional    elongation. Cell, 138(1): 129-45, 2009.-   16. Amir-Zilberstein, L.; Ainbinder, E.; Toube, L.; Yamaguchi, Y.;    Handa, H.; and Dikstein, R.: Differential regulation of NF-kappaB by    elongation factors is determined by core promoter type. Molecular    and cellular biology, 27(14): 5246-59, 2007.-   17. Brasier, A. R.: Expanding role of cyclin dependent kinases in    cytokine inducible gene expression. Cell cycle, 7(17): 2661-6, 2008.-   18. Barboric, M.; Nissen, R. M.; Kanazawa, S.; Jabrane-Ferrat, N.;    and Peterlin, B. M.: NF-kappaB binds P-TEFb to stimulate    transcriptional elongation by RNA polymerase II. Molecular Cell,    8(2): 327-37, 2001.-   19. Malumbres, M.; Pevarello, P.; Barbacid, M.; and Bischoff, J. R.:    CDK inhibitors in cancer therapy: what is next? Trends in    pharmacological sciences, 29(1): 16-21, 2008.-   20. Krystof, V., and Uldrijan, S.: Cyclin-dependent kinase    inhibitors as anticancer drugs. Current drug targets, 11(3):    291-302, 2010.-   21. Zhou, Q., and Yik, J. H.: The Yin and Yang of P-TEFb regulation:    implications for human immunodeficiency virus gene expression and    global control of cell growth and differentiation. Microbiology and    molecular biology reviews: MMBR, 70(3): 646-59, 2006.-   22. Rizzolio, F.; Tuccinardi, T.; Caligiuri, I.; Lucchetti, C.; and    Giordano, A.: CDK inhibitors: from the bench to clinical trials.    Current drug targets, 11(3): 279-90, 2010.-   23. Karaman, M. W. et al.: A quantitative analysis of kinase    inhibitor selectivity. Nature biotechnology, 26(1): 127-32, 2008.-   24. Phelps, M. A. et al.: Clinical response and pharmacokinetics    from a phase 1 study of an active dosing schedule of flavopiridol in    relapsed chronic lymphocytic leukemia. Blood, 113(12): 2637-45,    2009.-   25. Ni, W. et al.: Flavopiridol pharmacogenetics: clinical and    functional evidence for the role of SLCO1B1/OATP1B1 in flavopiridol    disposition. PLoS ONE, 5(11): e13792, 2010.-   26. Byrd, J. C. et al.: Flavopiridol administered using a    pharmacologically derived schedule is associated with marked    clinical efficacy in refractory, genetically high-risk chronic    lymphocytic leukemia. Blood, 109(2): 399-404, 2007.-   27. Sekine, C.; Sugihara, T.; Miyake, S.; Hirai, H.; Yoshida, M.;    Miyasaka, N.; and Kohsaka, H.: Successful treatment of animal models    of rheumatoid arthritis with small-molecule cyclin-dependent kinase    inhibitors. Journal of immunology, 180(3): 1954-61, 2008.-   28. Glasson, S. S. et al.: Deletion of active ADAMTSS prevents    cartilage degradation in a murinemodel of osteoarthritis. Nature,    434(7033): 644-8, 2005.-   29. Kamekura, S. et al.: Osteoarthritis development in novel    experimental mouse models induced byknee joint instability.    Osteoarthritis and cartilage/OARS, Osteoarthritis Research Society,    13(7): 632-41,2005.-   30. Glasson, S. S.; Blanchet, T. J.; and Morris, E. A.: The surgical    destabilization of the medialmeniscus (DMM) model of osteoarthritis    in the 129/SvEv mouse. Osteoarthritis and cartilage/OARS,    Osteoarthritis Research Society, 15(9): 1061-9, 2007.-   31. Poulet, B.; Hamilton, R. W.; Shefelbine, S.; and Pitsillides, A.    A.: Characterizing a novel and adjustable noninvasive murine joint    loading model. Arthritis & Rheumatism, 63(1): 137-147, 2011.-   32. van Osch, G. J.; van der Kraan, P. M.; Blankevoort, L.; Huiskes,    R.; and van den Berg, W. B.:Relation of ligament damage with site    specific cartilage loss and osteophyte formation incollagenase    induced osteoarthritis in mice. The Journal of rheumatology, 23(7):    1227-32, 1996.-   33. Joosten, L. A. et al.: Interleukin-18 promotes joint    inflammation and induces interleukin-l-driven cartilage destruction.    The American journal of pathology, 165(3): 959-67, 2004.-   34. Ameye, L. G., and Young, M. F.: Animal models of osteoarthritis:    lessons learned while seeking the““Holy Grail””. Curr Opin    Rheumatol, 18(5): 537-47, 2006.-   35. Dahlberg, L.; Roos, H.; Saxne, T.; Heinegard, D.; Lark, M. W.;    Hoerrner, L. A.; and Lohmander, L. S.: Cartilage metabolism in the    injured and uninjured knee of the same patient [see comments].    AnnRheum Dis, 53: 823-7, 1994.-   36. Glasson, S. S.; Chambers, M. G.; Van Den Berg, W. B.; and    Little, C. B.: The OARSI histopathology initiative - recommendations    for histological assessments of osteoarthritis in the mouse.    Osteoarthritis and cartilage/OARS, Osteoarthritis Research Society,    18 Suppl 3: S17-23, 2010.-   37. Aigner, T.; Cook, J. L.; Gerwin, N.; Glasson, S. S.; Laverty,    S.; Little, C. B.; McIlwraith, W.; and Kraus, V. B.: Histopathology    atlas of animal model systems—overview of guiding principles.    Osteoarthritis and cartilage/OARS, Osteoarthritis Research Society,    18 Suppl 3: S2-6, 2010.

Example 3 Inhibition of CDK9 Prevents Mechanical Injury-InducedInflammation, Apoptosis, and Matrix Degradation in Cartilage Explants

In this example, we examined the therapeutic potential of the CDK9inhibitor Flavopiridol in a single impact injury model with bovinecartilage explants. The ability of

Flavopiridol to prevent the activation of the injury-inducedinflammatory and catabolic responses, chondrocytes apoptosis, andcartilage matrix degradation was determined.

ABSTRACT

Joint injury often leads to post-traumatic osteoarthritis (PTOA). Acuteinjury responses to trauma induce production of pro-inflammatorycytokines and catabolic enzymes, which promote chondrocyte apoptosis anddegrade cartilage to potentiate POTA development. Recent studies showthat the rate-limiting step for transcriptional activation of injuryresponse genes is controlled by cyclin-dependent kinase 9 (CDK9), andthus it is an attractive target for limiting the injury response. Herewe determined the effects of CDK9 inhibition in suppressing the injuryresponse in mechanically-injured cartilage explants. Bovine cartilageexplants were injured by a single compressive load of 30% strain at100%/second, and then treated with the CDK9 inhibitor Flavopiridol. Toassess acute injury responses, we measured the mRNA expression ofpro-inflammatory cytokines, catabolic enzymes, and apoptotic genes byRT-PCR, and chondrocyte viability and apoptosis by TUNEL staining. Forlong-term outcome, cartilage matrix degradation was assessed by solubleglycosaminoglycan release, and by determining the mechanical propertieswith instantaneous and relaxation moduli. Our data showed CDK9 inhibitormarkedly reduced injury-induced inflammatory cytokine and catabolic geneexpression. CDK9 inhibitor also attenuated chondrocyte apoptosis andreduced cartilage matrix degradation. Lastly, the mechanical propertiesof the injured explants were preserved by

CDK9 inhibitor. Our results provide a temporal profile connecting thechain of events from mechanical impact, acute injury responses, to thesubsequent induction of chondrocyte apoptosis and cartilage matrixdeterioration. Thus, CDK9 is a potential disease modifying agent forinjury response after knee trauma to prevent or delay PTOA development.

MATERIALS AND METHODS

Cartilage Explants—

Bovine calf (˜2 months old, n=40 joints, sex unknown) stifle joints wereobtained from a local slaughterhouse (Petaluma, Calif.) within 1 day ofslaughtering. 6-8 cylindrical cartilage explants were harvested fromeach femoral condyle with a 6 mm biopsy punch inserted perpendicular tothe weight bearing area of the articular surface. The explants were thentrimmed into ˜3 mm thickness (with the articular surface intact and thedeep layer cut flat) using a custom jig. The explants were washed withphosphate buffered saline and cultured for 24 hours in high-glucoseDulbecco's Modified

Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum(Invitrogen), penicillin (1×10⁴ units/ml) and streptomycin (1×10⁴ μg/ml)at 37° C., 5% CO₂, and 95% relative humidity.

Single Impact Ex-Vivo Injury Model—

After a 24-hours recovery and equilibration period, the cartilageexplants were subjected to a single impact mechanical injury. Theprecise thickness of each individual explant was measured by a caliperbefore it was placed onto a custom-built unconfined loading chamber,with the articular surface facing upward. A 20 mm diameter stainlesssteel platen was lowered onto the explant surface to a pre-load of 0.5N(˜17.7 kPa) on a hydraulic material testing instrument (Instron8511.20). All explants including the uninjured controls were subjectedto this 0.5N pre-loading step. To avoid potential variation due to thepositional differences from where the explants were harvested on thecondyles, two adjacent explants were purposefully matched as a controland injured pair for later comparison. After pre-loading, the Instronwas programmed to deliver a single compression of 30% strain at100%/second, followed by immediate release. After the single impactloading, the explant was sliced in half and weighed. One half of theexplant was placed in 3 ml media and the other half placed in mediacontaining 300 nM Flavopiridol (Sigma) and cultured for various times(see FIG. 9A). The media was changed every other day with freshFlavopiridol added. The explants and the culturing media were thensubjected to further processing and analysis as described below.

Quantitative Real-Time PCR—

At 2-, 6-, and 24-hours post-injury, the explants were frozen in liquidnitrogen and pulverized with a pestle and mortar while frozen. Total RNAwas isolated with the miRNeasy Mini Kit (Qiagen) according to themanufacturer's instruction, with the exception that the RNA wasextracted twice with the Qiazol reagents to adequately remove thecartilage matrix constituents. The quantity and quality of the total RNAwere determined by a Nanodrop-2000 spectrophometer. 2.5 ug of total RNAfrom each sample was used for reverse transcription with the SuperScriptFirst-Strand RT kit (Invitrogen). Individual mRNA expression wasdetermined with quantitative real-time PCR performed in triplicates in a7900HT system (Applied Biosystem). Results were normalized to the 18srRNA (catalog no. 4319413E, Applied Biosystem) and calculated asfold-change in mRNA expression relative to control, using the 2^(−ΔΔCT)method. Probes used for individual bovine genes were custom made byIntegrated DNA Technologies.

Chondrocyte Viability—

The live and dead cells in the explants 5 days after mechanical injurywere stained using a Live/Dead Viability/Cytotoxicity kit (catalog no.L3224, Invitrogen) according to the manufacturer's protocol. Thepercentages of live and dead cells were determined by counting the cellnumbers in 3 random fields of the cross-sectional images of the explants(n=6 for each sample group) captured using a Nikon TE2000 invertedfluorescence microscope and a 20× objective.

Staining for Apoptotic Cells—

After 1, 3, and 5 days after injury, the explants were fixed with 4%paraformaldehyde for 24 hours and transferred to 75% ethanol, followedby sectioning for histological analysis. In situ detection of apoptosiswas performed on 5 um-thick whole explant cross-section using theDeadEnd Fluorometric TUNEL system kit (Promega). This kit measures thefragmented DNA of apoptotic cells by catalytically incorporatingfluorescein-12-dUTP at 3′-OH DNA ends using the TerminalDeoxynucleotidyl Transferase recombinant enzyme (rTdT). Nuclei werecounter stained with DAPI. The sections were mounted and examined undera fluorescence microscope. The percentage of apoptotic cells (n=3different donors) was determined by counting the number of TUNELpositive cells (green) and calculated as a percentage of the total cells(DAPI). Sections incubated with DNase I were used as positive controlwhile those incubated with buffer only were used as negative control.

GAG Release—

At 5-days post-injury, the culturing media was collected and the amountof glycosylaminoglycan (GAG) was determined by the dimethyl-methyleneblue (DMMB) colorimetric assay with chondroitin sulfate as the standard.Total GAG released into the medium was calculated and normalized to thewet weight of the explant (determined at the day of injury).

Cartilage Mechanical Properties—

To test if CDK9 inhibition preserves the mechanical properties ofcartilage explants after injury, injured and control explants werecultured for 4 weeks in media with or without Flavopiridol. Cartilagesample compressive properties were assessed by stress-relaxation testingin unconfined compression using a mechanical testing system (Instron5565, Norwood, Mass.). Prior to testing, a 3 mm diameter, 2 mm thickcompression sample was prepared using a dermal biopsy punch, then placedin phosphate buffered saline and centered beneath a 16 mm stainlesssteel platen. The platen was slowly lowered until a preload of 0.2N wasobserved, indicating contact between the platen and the cartilagesample. The sample was then preconditioned via fifteen cycles of 5%strain. All strains, including the preconditioning, were applied at astrain rate of 10% per second. Immediately following preconditioning,the sample was subject to 10% compressive strain; the 10% strain washeld constant and the load recorded for 380 seconds. At the end of the10% strain application, the compressive strain was increased to 20% andheld constant while the load was recorded for an additional 530 seconds.The compressive properties: instantaneous modulus, relaxation modulus,and coefficient of viscosity, were calculated from the individualstress-relaxation curves using data analysis software (MATLAB R2013a,Natick, Mass.) according to a standard linear solid model ofviscoelasticity as previously described (Allen and Athanasiou, 2006).This mechanical test and model were chosen for their simplicity andaccuracy in approximating the viscoelastic behavior of cartilage. Thecompressive properties of freshly isolated (day 0) bovine cartilageexplants from 6 donors were determined as baseline values for comparisonto 4-weeks post-injury samples.

Statistical Analysis—

Values of all measurements were expressed as the mean +/−standarddeviation. Changes in gene expression were analyzed by one-way ANOVAwith SPSS 16.0 software. The fold-change in mRNA was used as variablesto compare samples between different treatment groups. The leastsignificant difference post-hoc analysis was conducted with asignificance level of P<0.05.

Changes in compressive properties were analyzed by one-way ANOVA withTukey's post hoc test using JMP Pro software (version 11.2.0) with asignificance level of P<0.05.

RESULTS

CDK9 inhibition suppresses injury-induced pro-inflammatory and catabolicgenes—CDK9 controls the rate-limiting step of inflammatory geneactivation (Hargreaves et al., 2009; Zippo et al., 2009) and we havepreviously shown that in vitro CDK9 inhibition protects chondrocytes andcartilage from the catabolic effects of exogenously addedpro-inflammatory cytokines (Yik et al., 2014). However, the effects ofCDK9 inhibition on cartilage that receives a direct impact injury,similar to what may happen in a knee injury, have not been examined. Wehypothesize that CDK9 inhibition in mechanically injured cartilage willprevent an inflammatory response, which in turn will reduce thesubsequent deleterious effects on chondrocytes and the cartilage matrix.To test our hypothesis, bovine cartilage explants were mechanicallyinjured by subjecting them to an impact loading at a 30% strain rate(FIG. 9A). This magnitude of loading induces chondrocyte apoptosis andcartilage matrix degradation (Borrelli et al., 2003; D'Lima et al.,2001; Hembree et al., 2007; Loening et al., 2000; Morel and Quinn, 2004;Rosenzweig et al., 2012; Waters et al., 2014). The injured explants werecultured in the presence or absence of the CDK9 inhibitor Flavopiridolfor various times. The mRNA expression of inflammatory cytokines andcatabolic genes induced in the injured explants were determined andcompared to uninjured controls. The addition of Flavopiridol reduced theinduction of cytokine mRNA by injury (FIG. 9B&C, grey bars). This effectwas most pronounced in the expression of IL-6 mRNA, which wassignificantly induced by injury at all time points tested (FIG. 9B, openbars), and the IL-6 induction was markedly suppressed by Flavopiridol(FIG. 9B, grey bars). Similar trends were observed for IL-1β (FIG. 9C),although this did not reach statistical significance. These dataindicate that as expected, CDK9 inhibition in cartilage explantssuppresses inflammatory cytokine induction in response to mechanicalinjury.

We next examined the injured-induced changes in mRNA expression of thecatabolic genes MMP-1 and MMP-13, and ADAMTS4, which are induced byinflammatory cytokines and degrade the cartilage matrix. The resultsshowed that injury induced MMP-1 and ADAMTS4 expression significantly atall time points, and MMP-13 at the 2-hour time point (FIG. 9D-F, openbars). Inhibition of CDK9 effectively prevented the induction of thesegenes in the injured samples at all time points tested (FIG. 9D-F, greybars). In contrast, the mRNA expression of the anabolic genes Aggrecanand Col2a1 were not affected by injury whether or not Flavopiridol ispresent (FIG. 9G-H). Taken together, these results indicate that CDK9inhibition suppressed injury-induced catabolic mediators of cartilagematrix degradation, while the basal levels of anabolic genes are notaffected by injury or CDK9 inhibition at the time points tested.

CDK9 inhibition reduces injury-induced chondrocyte apoptosis—Besidesinducing an inflammatory response, mechanical injury also causeschondrocytes apoptosis in cartilage explants (Borrelli et al., 2003;D'Lima et al., 2001; Hembree et al., 2007; Rosenzweig et al., 2012), wetherefore investigated the effects of CDK9 inhibition on apoptosis usingour explant injury model. The mRNA expression of three selected genes(P53, Bcl-2 and PTEN) that are central to the apoptotic process wereexamined in the cartilage explants 24-hour post-injury. P53 initiatesapoptosis when DNA damage is irreparable (Amaral et al., 2010), Bcl-2 isthe founding member of anti-apoptotic factors (Czabotar et al., 2014)and PTEN is a crucial regulator of apoptosis (Zheng et al., 2010).Although mechanical injury itself did not significantly change the mRNAexpression of P53, Bcl-2, and PTEN (FIG. 10, open bars), Flavopiridolsignificantly decreased the expression of these apoptotic mediators inboth the injured and uninjured groups (FIG. 10, grey bars).

The reduced basal level of apoptotic mediators has prompted us todirectly determine the number of apoptotic chondrocytes in the injuredcartilage explants. Cartilage explants from three different donorscollected at 1-, 3-, and 5-day post-injury were examined by TUNEL stainand the percentage of apoptotic cells relative to the total number ofnuclei in each sample was determined. The results showed that injuryincreased the percentage of apoptotic chondrocytes to ˜20% at 1-daypost-injury, and to ˜60% 3 days after injury (FIG. 11A). In contrast,CDK9 inhibition significantly reduced the percentage of apoptotic cellsin the injured explants in all time points tested (FIG. 11A). Theseresults were further corroborated by the data on live/dead staining ofcartilage explants collected 5-day post-injury (FIG. 11B). The live/deadstain showed that injury significantly reduced the number of livechondrocytes from ˜80% to ˜55% (FIG. 11B, open bars). Flavopiridoltreatment enhanced cell survival in the injured explants and did notsignificantly affect cell survival in uninjured explants (FIG. 11B, greybars). Taken together, the above results indicate that CDK9 inhibitiondecreases injury-induced apoptosis in cartilage explants and enhancechondrocyte survival after impact injury.

CDK9 inhibition prevents injury-induced cartilage matrixdegradation—Since injury-induced catabolic response leads toupregulation of matrix degrading enzymes, we next investigated if CDK9inhibition could prevent cartilage matrix degradation after injury. Thecartilage explants were continuously cultured for 5 days in the presenceor absence of 300 nM Flavopiridol after injury, the culturing media wascollected and the GAG content released into the media was determined asa measure of matrix degradation. GAG release was significantly increasedupon injury (FIG. 12, open bars); however, in samples treated withFlavopiridol, GAG release by injured cartilage was not increased abovethe uninjured control baseline. This result indicates that the CDK9inhibition attenuates mechanical injury-induced cartilage matrixdegradation and proteoglycan release.

CDK9 inhibition preserves the mechanical properties of cartilageexplants after injury−The compressive properties of freshly isolatedcartilage were determined from 6 individual donors and their mean valueswere indicated by the dotted lines in FIG. 13. The compressiveproperties of the cartilage explants following four weeks of culturedemonstrate increased 10% relaxation modulus, 10% instantaneous modulus,and 10% coefficient of viscosity in samples treated with Flavopiridol,when compared to injured, untreated samples (FIG. 13). Similar resultswere observed for the moduli and coefficients of viscosity calculatedfrom the 20% strain stress-relaxation curves (not shown). The positiveeffect of Flavopiridol on compressive properties was observed in bothinjured and uninjured cartilage samples; no significant difference wasobserved for any compressive property between injured and uninjuredcartilage samples when treated with Flavopiridol. Although injured,untreated cartilage samples exhibited the lowest compressive propertiesamong all groups, there was no significant difference between injuredand uninjured cartilage samples in the absence of Flavopiridol.Interestingly, cartilage samples treated with Flavopiridol exhibitedgreater relaxation moduli than untreated, uninjured controls.Furthermore, injured cartilage samples treated with Flavopiridol alsohave greater instantaneous moduli and coefficients of viscosity thanuntreated, uninjured controls. These results indicate that Flavopiridolhas a beneficial effect on the compressive properties of cartilagesamples cultured in vitro.

DISCUSSION

This study examined the effects of CDK9 inhibition on the biological andmechanical properties of cartilage explants after injury by compressiveloading. This injury model caused a significant induction ofpro-inflammatory cytokines and catabolic enzymes (FIG. 9) within thefirst 24 hours. Although apoptotic genes were unchanged within thisperiod (FIG. 10), apoptotic chondrocytes could be detected in theexplants at 1 day following injury and peaked after 5 days (FIG. 11).Injury also accelerated cartilage matrix degradation as measured byGAGrelease (FIG. 12). However, CDK9 inhibition by Flavopiridol suppressedall those changes and preserved the mechanical properties similar tothose of native cartilage (FIG. 13), thus effectively protecting thechondrocytes and the cartilage from the harmful effects of physicalinjury and the inflammatory response that follows.

A high incidence of OA is associated with traumatic knee injury andhence the term post-traumatic OA (PTOA). Although the pathology of thedevelopment of PTOA from injury remains unclear, several lines ofevidence point to the involvement of the inflammatory response in thisprocess. Elevated levels of the inflammatory cytokines IL-1β and IL-6are detected in the human joints within 24 hours after an ACL injury(Irie et al., 2003). Ko et al demonstrate that inflammation and theaccompanying dysregulated cytokines activities likely contribute to thedisruption of the balance between anabolism and catabolism in OA(Goldring and Berenbaum, 2004). In vitro and in vivo studies haveimplicated pro-inflammatory cytokines, particularly IL-1β, in thedestruction of articular cartilage in OA (Goldring and Berenbaum, 2004;Kobayashi et al., 2005). In cartilage, chondrocytes are the main targetof pro-inflammatory cytokines, which dysregulate the expression ofcatabolic and anabolic genes. Cytokine-stimulated chondrocytes produce avariety of matrix-degrading enzymes, like MMP-1, -3, -13 and theaggrecanase ADAMTS-4, -5 (Lee et al., 2005; Nishimuta and Levenston,2012). All these data implicate the involvement of the inflammatoryresponse in cartilage matrix degradation. Our results in this studystrongly indicate that CDK9 inhibition prevents induction ofinflammatory and catabolic response genes and thus is a novel target forpreventing injury-induced damage.

The influences of mechanical injury on chondrocytes anabolic geneexpression are still controversial. It has been reported that IL-1βsuppresses the expression of Col2a1 in chondrocytes in vitro (Okazaki etal., 2002). However, other OA studies show that in osteoarthriticcartilage, there is enhanced aggrecan and Col2a1 gene expression andbiosynthesis, when compared to normal cartilage (Bau et al., 2002;Hermansson et al., 2004). Our results showed no significant change ofanabolic gene expression in injured cartilage in the first 24 hours.This is supported by large-scale expression profiling studies usefull-thickness cartilage, which demonstrated that many anabolic genes,including Col2a1, are only enhanced in late-stage OA (Aigner et al.,2006; Ijiri et al., 2008). More importantly, our result demonstratesthat Flavopiridol has no adverse side effects on anabolic geneexpression in the first 24 hours after mechanical injury.

Mechanical injury to cartilage leads to chondrocyte apoptosis. D'Lima etal applied a 30% strain to injure cartilage explants and found 34%apoptotic chondrocytes in 96 hours, compare to only 4% apoptosis in thecontrol group (D′Lima et al., 2001). Similarly, our explant injury modelalso leads to chondrocyte apoptosis, we detect ˜20% apoptoticchondrocytes in 24 hours and more than 60% apoptotic cells after 72hours (FIG. 11A). Given that injury to cartilage and chondrocyteapoptosis lead to cartilage degradation, the development of drugsdesigned to block this could be beneficial in preventing PTOAdevelopment (Borrelli et al., 2003; D'Lima et al., 2001). For example,when mechanically injured cartilage explants was treated with caspaseinhibitors, a 50% reduction of apoptosis was seen (D'Lima et al., 2001).However, few studies have focused on the correlation between the acuteinjury response within hours, such as induction of inflammatory cytokineand catabolic genes, and the subsequent apoptosis that followed, usuallyat several days post-injury. Pro-inflammatory cytokines like IL-1β andIL-6 have the capacity to activate a diverse array of intracellularsignaling pathways, such as JNK, p38 MAPK and NF-kB (Goldring et al.,2011), and further induce the expression of various pro-apoptotic geneslike p53 (Amaral et al., 2010), BCL-2 (Czabotar et al., 2014) and PTEN(Zheng et al., 2010). In chondrocytes, JNK and p38 signaling pathwaysare thought to be pro-apoptotic in an injury response (Rosenzweig etal., 2012). Results from this study provide a temporal profileconnecting the chain of events that happen after mechanical injury tocartilage, from the induction of inflammatory cytokines and catabolicgenes, to the subsequent induction of chondrocyte apoptosis.Importantly, Flavopiridol treatment effectively blocks the initial phaseof the injury response, and thus prevents subsequent damage to thecartilage.

In contrast to the anti-apoptotic property in this study, Flavopiridolhas been reported to induce apoptosis in many cancer cells. Notably,Flavopiridol was originally known for its anti-proliferation propertiesby suppressing cell-cycle progression in rapidly dividing cells (e.g.cancers) (Wang and Ren, 2010). This is due to the off-target suppressiveeffect of Flavopiridol on other CDKs that directly regulate cell-cycle.We believe that mature chondrocytes, which do not normally divide(Kobayashi et al., 2005), are therefore less sensitive to theanti-proliferative effect of Flavopiridol.

Flavopiridol has a beneficial effect on the compressive, viscoelasticproperties of injured cartilage explants after four weeks of culture.Specifically, both injured and uninjured cartilage samples treated withFlavopiridol have increased stiffness upon initial compression(instantaneous modulus) and upon reaching equilibrium during prolongedstatic compression (relaxation modulus) compared to injured-untreatedsamples. The increased instantaneous and relaxation moduli are likelyassociated with preservation of glycosaminoglycans (GAG) in thecartilage extracellular matrix (ECM) and of the elastic stiffness of theECM, respectively. Furthermore, injured samples treated withFlavopiridol exhibit a slower rate of relaxation toward equilibriumfollowing compression compared to injured-untreated samples as evidencedby a greater coefficient of viscosity in the treated samples. Anincreased coefficient of viscosity is likely associated with retentionof GAG in the ECM, which resists movement of water out of cartilageduring compression. Overall, treatment with Flavopiridol helps maintainthe viscoelastic, compressive properties of cultured cartilage samplesclose to values observed for uncultured native cartilage samples (FIG.13). Flavopiridol may preserve cartilage mechanical properties in vivofollowing injury as well as in vitro during culture.

The lack of difference in the mechanical properties between injuredcartilage explants and uninjured control explants was an unexpectedfinding of this study. Although the injured explants cultured withoutFlavopiridol had the lowest instantaneous modulus and coefficient ofviscosity among all treatment groups, the difference between injured anduninjured samples was not statistically significant (P>0.05). Cartilagedegradation and loss of GAG known to occur during prolonged culture ofimmature cartilage (Bian et al., 2010) may have caused the loss ofcartilage compressive properties observed in the untreated-uninjuredcartilage explants. We suspect that testing samples after shorterculture duration may demonstrate a greater difference between theinjured and uninjured cartilage.

In summary, out data for the first time demonstrated the effectivenessof CDK9 inhibition in the suppression of pro-inflammatory cytokineinduced by mechanical injury and prevention of chondrocyte apoptosis incartilage explants. In addition, our data strongly indicate thatFlavopiridol is an effective agent to prevent cartilage matrixdegradation and to preserve its mechanical properties after injury. ThusCDK9 inhibition by Flavopiridol provides an effective strategy toprevent or delay PTOA after knee joint trauma.

REFERENCES FOR EXAMPLE 3

Aigner T, Fundel K, Saas J, Gebhard P M, Haag J, Weiss T, Zien A,Obermayr F, Zimmer R, Bartnik E (2006) Large-scale gene expressionprofiling reveals major pathogenetic pathways of cartilage degenerationin osteoarthritis. Arthritis Rheum 54: 3533-3544.

Allen K D, Athanasiou K A (2006) Viscoelastic characterization of theporcine temporomandibular joint disc under unconfined compression. JBiomech 39: 312-322.

Amaral J D, Xavier J M, Steer C J, Rodrigues C M (2010) The role of p53in apoptosis. Discov Med 9: 145-152.

Bau B, Haag J, Schmid E, Kaiser M, Gebhard P M, Aigner T (2002) Bonemorphogenetic protein-mediating receptor-associated Smads as well ascommon Smad are expressed in human articular chondrocytes but notup-regulated or down-regulated in osteoarthritic cartilage. J Bone MinerRes 17: 2141-2150.

Bian L, Stoker A M, Marberry K M, Ateshian G A, Cook J L, Hung C T(2010) Effects of dexamethasone on the functional properties ofcartilage explants during long-term culture. Am J Sports Med 38: 78-85.

Blumberg T J, Natoli R M, Athanasiou K A (2008) Effects of doxycyclineon articular cartilage GAG release and mechanical properties followingimpact. Biotechnol Bioeng 100: 506-515.

Borrelli J, Jr., Tinsley K, Ricci W M, Burns M, Karl I E, Hotchkiss R(2003) Induction of chondrocyte apoptosis following impact load. JOrthop Trauma 17: 635-641.

Christiansen B A, Anderson M J, Lee C A, Williams J C, Yik J H,Haudenschild D R (2012) Musculoskeletal changes following non-invasiveknee injury using a novel mouse model of post-traumatic osteoarthritis.Osteoarthritis Cartilage 20: 773-782.

Czabotar P E, Lessene G, Strasser A, Adams J M (2014) Control ofapoptosis by the BCL-2 protein family: implications for physiology andtherapy. Nat Rev Mol Cell Biol 15: 49-63.

D'Lima DD, Hashimoto S, Chen P C, Colwell C W, Jr., Lotz M K (2001)Human chondrocyte apoptosis in response to mechanical injury.Osteoarthritis Cartilage 9: 712-719.

Fowler T, Sen R, Roy AL (2011) Regulation of primary response genes. MolCell 44: 348-360.

Goldring M B, Berenbaum F (2004) The regulation of chondrocyte functionby proinflammatory mediators: prostaglandins and nitric oxide. ClinOrthop Relat Res: S37-46.

Goldring M B, Otero M, Plumb D A, Dragomir C, Favero M, El Hachem K,Hashimoto K, Roach H I, Olivotto E, Borzi R M, Marcu K B (2011) Roles ofinflammatory and anabolic cytokines in cartilage metabolism: signals andmultiple effectors converge upon MMP-13 regulation in osteoarthritis.Eur Cell Mater 21: 202-220.

Guilak F, Fermor B, Keefe F J, Kraus V B, Olson S A, Pisetsky D S,Setton L A, Weinberg J B (2004) The role of biomechanics andinflammation in cartilage injury and repair. Clin Orthop Relat Res:17-26.

Hargreaves D C, Horng T, Medzhitov R (2009) Control of inducible geneexpression by signal-dependent transcriptional elongation. Cell 138:129-145.

Hembree W C, Ward B D, Furman B D, Zura R D, Nichols L A, Guilak F,Olson S A (2007) Viability and apoptosis of human chondrocytes inosteochondral fragments following joint trauma. J Bone Joint Surg Br 89:1388-1395.

Hermansson M, Sawaji Y, Bolton M, Alexander S, Wallace A, Begum S, WaitR, Saklatvala J (2004) Proteomic analysis of articular cartilage showsincreased type II collagen synthesis in osteoarthritis and expression ofinhibin betaA (activin A), a regulatory molecule for chondrocytes. JBiol Chem 279: 43514-43521.

Ijiri K, Zerbini L F, Peng H, Otu H H, Tsuchimochi K, Otero M, DragomirC, Walsh N, Bierbaum B E, Mattingly D, van Flandern G, Komiya S, AignerT, Libermann T A, Goldring M B (2008) Differential expression ofGADD45beta in normal and osteoarthritic cartilage: potential role inhomeostasis of articular chondrocytes. Arthritis Rheum 58: 2075-2087.

Imgenberg J, Rolauffs B, Grodzinsky A J, Schunke M, Kurz B (2013)Estrogen reduces mechanical injury-related cell death and proteoglycandegradation in mature articular cartilage independent of the presence ofthe superficial zone tissue. Osteoarthritis Cartilage 21: 1738-1745.

Irie K, Uchiyama E, Iwaso H (2003) Intraarticular inflammatory cytokinesin acute anterior cruciate ligament injured knee. Knee 10: 93-96.

Ko F C, Dragomir C, Plumb D A, Goldring S R, Wright T M, Goldring M B,van der Meulen M C (2013) In vivo cyclic compression causes cartilagedegeneration and subchondral bone changes in mouse tibiae. ArthritisRheum 65: 1569-1578.

Kobayashi M, Squires G R, Mousa A, Tanzer M, Zukor D J, Antoniou J,Feige U, Poole A R (2005) Role of interleukin-1 and tumor necrosisfactor alpha in matrix degradation of human osteoarthritic cartilage.Arthritis Rheum 52: 128-135.

Lee J H, Fitzgerald J B, Dimicco M A, Grodzinsky A J (2005) Mechanicalinjury of cartilage explants causes specific time-dependent changes inchondrocyte gene expression. Arthritis Rheum 52: 2386-2395.

Lockwood K A, Chu B T, Anderson M J, Haudenschild D R, Christiansen B A(2014) Comparison of loading rate-dependent injury modes in a murinemodel of post-traumatic osteoarthritis. J Orthop Res 32: 79-88.

Loening A M, James I E, Levenston M E, Badger A M, Frank E H, Kurz B,Nuttall M E, Hung H H, Blake S M, Grodzinsky A J, Lark M W (2000)Injurious mechanical compression of bovine articular cartilage induceschondrocyte apoptosis. Arch Biochem Biophys 381: 205-212.

Lohmander L S, Englund P M, Dahl L L, Roos E M (2007) The long-termconsequence of anterior cruciate ligament and meniscus injuries:osteoarthritis. Am J Sports Med 35: 1756-1769.

Morel V, Quinn T M (2004) Cartilage injury by ramp compression near thegel diffusion rate. J Orthop Res 22: 145-151.

Murray M M, Zurakowski D, Vrahas M S (2004) The death of articularchondrocytes after intra-articular fracture in humans. J Trauma 56:128-131.

Nishimuta J F, Levenston M E (2012) Response of cartilage and meniscustissue explants to in vitro compressive overload. OsteoarthritisCartilage 20: 422-429.

Okazaki K, Li J, Yu H, Fukui N, Sandell L J (2002)CCAAT/enhancer-binding proteins beta and delta mediate the repression ofgene transcription of cartilage-derived retinoic acid-sensitive proteininduced by interleukin-1 beta. J Biol Chem 277: 31526-31533.

Rosenzweig D H, Djap M J, Ou S J, Quinn T M (2012) Mechanical injury ofbovine cartilage explants induces depth-dependent, transient changes inMAP kinase activity associated with apoptosis. Osteoarthritis Cartilage20: 1591-1602.

Wang L M, Ren D M (2010) Flavopiridol, the first cyclin-dependent kinaseinhibitor: recent advances in combination chemotherapy. Mini Rev MedChem 10: 1058-1070.

Waters N P, Stoker A M, Carson W L, Pfeiffer F M, Cook J L (2014)Biomarkers affected by impact velocity and maximum strain of cartilageduring injury. J Biomech 47: 3185-3195.

Yik JH, Hu Z, Kumari R, Christiansen BA, Haudenschild DR (2014)Cyclin-dependent kinase 9 inhibition protects cartilage from thecatabolic effects of proinflammatory cytokines. Arthritis Rheumatol 66:1537-1546.

Zheng T, Meng X, Wang J, Chen X, Yin D, Liang Y, Song X, Pan S, Jiang H,Liu L (2010) PTEN- and p53-mediated apoptosis and cell cycle arrest byFTY720 in gastric cancer cells and nude mice. Journal of CellularBiochemistry 111: 218-228.

Zhou Q, Yik JH (2006) The Yin and Yang of P-TEFb regulation:implications for human immunodeficiency virus gene expression and globalcontrol of cell growth and differentiation. Microbiol Mol Biol Rev 70:646-659.

Zippo A, Serafini R, Rocchigiani M, Pennacchini S, Krepelova A, OlivieroS (2009) Histone crosstalk between H3S10ph and H4K16ac generates ahistone code that mediates transcription elongation. Cell 138:1122-1136.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of reducing, preventing, inhibiting,mitigating and/or ameliorating cartilage degradation and/or chondrocytedeath in a subject in need thereof, comprising co-administering to thesubject an effective amount of an inhibitor of cyclin-dependent kinase 9(CDK9) and an inhibitor of bromodomain containing 4 (BRD4), therebyreducing, preventing, inhibiting, mitigating and/or amelioratingcartilage degradation and/or chondrocyte death in the subject.
 2. Amethod of reducing, preventing, inhibiting, mitigating and/orameliorating the onset and/or progression of post-traumaticosteoarthritis in a subject in need thereof, comprising co-administeringto the subject an effective amount of an inhibitor of cyclin-dependentkinase 9 (CDK9) and an inhibitor of bromodomain containing 4 (BRD4),thereby reducing, preventing, inhibiting, mitigating and/or amelioratingpost-traumatic osteoarthritis in the subject.
 3. The method of any oneof claims 1 to 2, wherein the subject has experienced a traumatic injuryto cartilage tissue.
 4. The method of claim 3, wherein the subject hasundergone joint surgery.
 5. A method of reducing, preventing,inhibiting, mitigating and/or ameliorating severe polytrauma or systemicinflammatory response syndrome (SIRS) in a subject in need thereof,comprising administering to the subject an effective amount of aninhibitor of cyclin-dependent kinase 9 (CDK9), thereby reducing,preventing, inhibiting, mitigating and/or ameliorating severe polytraumaor systemic inflammatory response syndrome (SIRS) in the subject.
 6. Amethod of reducing, preventing, inhibiting, mitigating and/orameliorating severe polytrauma or systemic inflammatory responsesyndrome (SIRS) in a subject in need thereof, comprisingco-administering to the subject an effective amount of an inhibitor ofcyclin-dependent kinase 9 (CDK9) and an inhibitor of bromodomaincontaining 4 (BRD4), thereby reducing, preventing, inhibiting,mitigating and/or ameliorating severe polytrauma or systemicinflammatory response syndrome (SIRS) in the subject.
 7. The method ofany one of claims 1 to 6, wherein one or both of the inhibitor of CDK9and the inhibitor of BRD4 are initially co-administered during the acuteresponse phase and reduce and/or inhibit transcriptional activation ofone or more primary response genes after experiencing traumatic injury.8. The method of claim 7, wherein the one or more primary response genesare selected from the group consisting of IL-1β, inducible nitric oxidesynthase (iNOS), IL-6, TNF-α, MMP-1, MMP-3, MMP-9, MMP-13 and ADAMTS4(aggrecanase).
 9. The method of any one of claims 1 to 8, wherein one orboth of the inhibitor of CDK9 and the inhibitor of BRD4 are initiallyco-administered within about 72 hours after experiencing traumaticinjury.
 10. The method of any one of claims 1 to 6, wherein one or bothof the inhibitor of CDK9 and the inhibitor of BRD4 are administered tothe subject within about 10 days after damage or injury.
 11. The methodof any one of claims 1 to 6, wherein one or both of the inhibitor ofCDK9 and the inhibitor of BRD4 are administered over a course of 10days.
 12. The method of any one of claims 1 to 11, wherein one or bothof the inhibitor of CDK9 and the inhibitor of BRD4 are administeredmultiple times.
 13. The method of any one of claims 1 to 12, wherein thesubject has undergone surgery to repair damaged cartilage tissue. 14.The method of any one of claims 1 to 13, wherein the subject hasreceived an osteochondral explant.
 15. The method of claim 14, whereinthe osteochondral explant is a cartilage allograft.
 16. The method ofany one of claims 1 to 15, wherein one or both of the inhibitor of CDK9and the inhibitor of BRD4 are co-administered concurrently with or priorto surgery.
 17. The method of any one of claims 1 to 15, wherein one orboth of the inhibitor of CDK9 and the inhibitor of BRD4 areco-administered within 24 hours after surgery.
 18. The method of any oneof claims 1 to 17, wherein one or both of the inhibitor of CDK9 and theinhibitor of BRD4 are co-administered systemically.
 19. The method ofclaim 18, wherein one or both of the inhibitor of CDK9 and the inhibitorof BRD4 are co-administered intravenously.
 20. The method of any one ofclaims 1 to 17, wherein one or both of the inhibitor of CDK9 and theinhibitor of BRD4 are co-administered directly to the site of injuredcartilage tissue.
 21. The method of claim 20, wherein one or both of theinhibitor of CDK9 and the inhibitor of BRD4 are delivered from a matrix.22. The method of any one of claims 1 to 20, wherein the inhibitor ofCDK9 is a small organic compound.
 23. The method of claim 22, whereinthe inhibitor of CDK9 is flavopiridol.
 24. The method of any one ofclaims 1 to 23, wherein the inhibitor of BRD4 is a small organiccompound.
 25. The method of claim 24, wherein the inhibitor of BRD4 isselected from the group consisting of JQ1 and GSK525762A.
 26. The methodof any one of claims 1 to 25, wherein one or both of the inhibitor ofCDK9 and the inhibitor of BRD4 are administered at a subtherapeutic ornon-efficacious dose for the individual inhibitors.
 27. A method ofreducing, preventing or inhibiting degradation of an osteochondralexplant and/or reducing, preventing or inhibiting chondrocyte deathduring storage, comprising storing the osteochondral explant and/orchondrocytes in a solution comprising an inhibitor of cyclin-dependentkinase 9 (CDK9) and an inhibitor of bromodomain containing 4 (BRD4). 28.The method of claim 27, wherein the osteochondral explant is allograftcartilage.
 29. The method of any one of claims 27 to 28, wherein theinhibitor of CDK9 is a small organic compound.
 30. The method of any oneof claims 27 to 29, wherein the inhibitor of CDK9 is flavopiridol. 31.The method of any one of claims 27 to 30, wherein the inhibitor of BRD4is a small organic compound.
 32. The method of any one of claims 27 to31, wherein the inhibitor of BRD4 is selected from JQ1 and GSK525762A.33. The method of any one of claims 27 to 32, wherein the osteochondralexplant is submerged in the solution comprising the inhibitor of CDK9and the inhibitor of BRD4.
 34. The method of any one of claims 27 to 33,wherein the solution comprises a sub-efficacious amount of one or bothof the inhibitor of CDK9 and the inhibitor of BRD4.
 35. A compositioncomprising an osteochondral explant in a solution comprising aninhibitor of cyclin-dependent kinase 9 (CDK9) and an inhibitor ofbromodomain containing 4 (BRD4).
 36. The composition of claim 35,wherein the osteochondral explant is allograft cartilage.
 37. Thecomposition of any one of claims 35 to 36, wherein the inhibitor of CDK9is a small organic compound.
 38. The composition of any one of claims 35to 37, wherein the inhibitor of CDK9 is flavopiridol.
 39. Thecomposition of any one of claims 35 to 38, wherein the inhibitor of BRD4is a small organic compound.
 40. The composition of any one of claims 35to 39, wherein the inhibitor of BRD4 is selected from JQ1 andGSK525762A.
 41. The composition of any one of claims 35 to 40, whereinthe osteochondral explant is submerged in the solution comprising theinhibitor of CDK9 and the inhibitor of BRD4.
 42. The composition of anyone of claims 35 to 41, wherein the solution comprises a sub-efficaciousamount of one or both of the inhibitor of CDK9 and the inhibitor ofBRD4.
 43. A kit comprising the composition of any one of claims 35 to42.